Antibodies reactive with human carcinomas

ABSTRACT

The present invention relates to novel antibodies, antibody fragments and antibody conjugates and single-chain immunotoxins reactive with human carcinoma cells. More particularly, the antibodies, conjugates and single-chain immunotoxins of the invention include: a murine monoclonal antibody, BR96; a human/murine chimeric antibody, ChiBR96; a F(ab&#39;) 2  fragment of BR96; ChiBR96-PE, ChiBR96-LysPE40, ChiBR96 F(ab&#39;) 2  -LysPE40 and ChiBR96 Fab&#39;-LysPE40 conjugates and recombinant BR96 sFv-PE40 immunotoxin. These molecules are reactive with a cell membrane antigen on the surface of human carcinomas. The BR96 antibody and its functional equivalents, displays a high degree of selectivity for carcinoma cells and possess the ability to mediate antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity activity. In addition, the antibodies of the invention internalize within the carcinoma cells to which they bind and are therefore particularly useful for therapeutic applications, for example, as the antibody component of antibody-drug or antibody-toxin conjugates. The antibodies also have a unique feature in that they are cytotoxic when used in the unmodified form, at specified concentrations.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Ser. No. 07/892,501,filed Jun. 1, 1992, now abandoned, which is a continuation-in-part ofU.S. Ser. No. 07/544,246, filed Jun. 26, 1990, now abandoned, which is acontinuation-in-part of Ser. No. 07/374,947, filed Jun. 30, 1989, nowabandoned. In addition, this application is a continuation-in-part ofU.S. Ser. No. 08/057,444, filed May 5, 1993, now U.S. Pat. No.5,491,088, which is a continuation of U.S. Ser. No. 07/544,246, filedJun. 26, 1990, now abandoned, which is a continuation-in-part of U.S.Ser. No. 07/374,947, filed Jun. 30, 1989, now abandoned. The entiredisclosures of these applications are hereby incorporated by reference.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to novel antibodies reactive withcarcinoma cells. More particularly, the invention relates to a murinemonoclonal antibody and a chimeric monoclonal antibody, includingimmunoconjugates and recombinant immunotoxins made therefrom, that reactwith cell membrane antigens associated with a large variety ofcarcinomas including carcinomas of the colon, breast, ovary and lung.The murine monoclonal antibody is highly specific for carcinomas,showing no to very low reactivity with normal animal tissues or othertypes of tumors such as lymphomas or sarcomas.

BACKGROUND OF THE INVENTION

1. Monoclonal Antibodies Directed Against Cell Membrane Antigens.

Monoclonal antibodies (MAbs) to human tumor-associated differentiationantigens offer promises for the "targeting" of various antitumor agentssuch as radioisotopes, chemotherapeutic drugs, and toxins. [Order, in"Monoclonal Antibodies for Cancer Detection and Therapy", Baldwin andByers, (eds.),London, Academic Press (1985)].

In addition, some monoclonal antibodies have the advantage of killingtumor cells via antibody-dependent cellular cytotoxicity (ADCC) orcomplement-dependent cytotoxicity (CDC) in the presence of humaneffector cells or serum [Hellstrom et al., Proc. Natl. Acad. Sci. USA83:7059-7063 (1986)], and there are a few monoclonal antibodies thathave a direct antitumor activity which does not depend on any hostcomponent [Drebin et al., Oncogene 2:387-394 (1988)].

Many monoclonal antibodies reactive with carcinoma-associated antigensare known [see, e.g., Papsidero, "Recent Progress In The ImmunologicalMonitoring Of Carcinomas Using Monoclonal Antibodies", Semin. Surg.Oncol., 1 (4):171-81 (1985); Schlom et al., "Potential Clinical UtilityOf Monoclonal Antibodies In The Management Of Human Carcinomas",Important Adv. Oncol., 170-92 (1985); Allum et al., "MonoclonalAntibodies In The Diagnosis And Treatment of Malignant Conditions",Surg. Ann., 18:41-64 (1986); and Houghton et al., "MonoclonalAntibodies: Potential Applications To The Treatment Of Cancer", Semin.Oncol., 13(2):165-79 (1986)].

These known monoclonal antibodies can bind to a variety of differentcarcinoma-associated antigens including glycoproteins, glycolipids andmucins [see, e.g., Fink et al., "Monoclonal Antibodies As DiagnosticReagents for The Identification And Characterization Of Human TumorAntigens", Prog. Clin. Pathol., 9:121-33 (1984)].

For example, monoclonal antibodies that bind to glycoprotein antigens onspecific types of carcinomas include those described in U.S. Pat. No.4,737,579 (monoclonal antibodies to non-small cell lung carcinomas),U.S. Pat. No. 4,753,894 (monoclonal antibodies to human breast cancer),U.S. Pat. No. 4,579,827 (monoclonal antibodies to human gastrointestinalcancer), and U.S. Pat. No. 4,713,352 (monoclonal antibodies to humanrenal carcinoma).

Monoclonal antibody B72.3, which is one of the antibodies studied themost, recognizes a tumor-associated mucin antigen of greater than 1,000kd molecular weight that is selectively expressed on a number ofdifferent carcinomas. Thus, B72.3 has been shown to react with 84% ofbreast carcinomas, 94% of colon carcinomas, 100% of ovarian carcinomasand 96% of non-small cell lung carcinomas [see Johnston, "Applicationsof Monoclonal Antibodies In Clinical Cytology As Exemplified By StudiesWith Monoclonal Antibody B72.3", Acta Cytol., 1(5): 537-56 (1987) andU.S. Pat. No. 4,612,282, issued to Schlom et al.]. Another patentedmonoclonal antibody, KC-4, [see U.S. Pat. No. 4,708,930], recognizes anapproximately 400-500 kd protein antigen expressed on a number ofcarcinomas, such as colon, prostate, lung and breast carcinoma. Itappears that neither the B72.3 nor KC-4 antibodies internalize withinthe carcinoma cells with which they react.

Monoclonal antibodies reactive with glycolipid antigens associated withtumor cells have been disclosed. For example, Young et al., "ProductionOf Monoclonal Antibodies Specific For Two Distinct Steric Portions OfThe Glycolipid Ganglio-N-Triosylceramide (Asialo GM₂)", J. Exp. Med.,150: 1008-1019 (1979) disclose the production of two monoclonalantibodies specific for asialo GM₂, a cell surface glycosphingolipidantigen that was established as a marker for BALB/c V3T3 cellstransformed by Kirsten murine sarcoma virus. See, also, Kniep et al.,"Gangliotriasylceramide (Asialo GM₂) A Glycosphingolipid Marker For CellLines Derived From Patients With Hodgkin's Disease", J. Immunol.,131(3): 1591-94 (1983) and U.S. Pat. No. 4,507,391 (monoclonal antibodyto human melanoma).

Other monoclonal antibodies reactive with glycolipid antigens oncarcinoma cells include those described by Rosen et al., "Analysis OfHuman Small Cell Lung Cancer Differentiation Antigens Using A Panel OfRat Monoclonal Antibodies", Cancer Research, 44:2052-61 (1984)(monoclonal antibodies to human small cell lung cancer), Varki et al.,"Antigens Associated with a Human Lung Adenocarcinoma Defined byMonoclonal Antibodies", Cancer Research 44:681-87 (1984); (monoclonalantibodies to human adenocarcinomas of the lung, stomach and colon andmelanoma), and U.S. Pat. No. 4,579,827 (monoclonal antibodies to humancolon adenocarcinoma). See, also, Hellstrom et al., "Antitumor EffectsOf L6, An IgG2a Antibody That Reacts With Most Human Carcinomas", Proc.Natl. Acad. Sci. USA, 83:7059-63 (1986) which describes the L6monoclonal antibody that recognizes a carbohydrate antigen expressed onthe surface of human non-small cell lung carcinomas, breast carcinomasand colon carcinomas.

Antibodies to tumor-associated antigens which are not able tointernalize within the tumor cells to which they bind are generally notuseful to prepare conjugates with antitumor drugs or toxins, since thesewould not be able to reach their site of action within the cell. Otherapproaches would then be needed so as to use such antibodiestherapeutically.

Additional monoclonal antibodies exhibiting a high specific reactivityto the majority of cells from a wide range of carcinomas are greatlyneeded. This is so because of the antigenic heterogeneity of manycarcinomas which often necessitates, in diagnosis or therapy, the use ofa number of different monoclonal antibodies to the same tumor mass.There is a further need, especially for therapy, for so called"internalizing" antibodies, i.e., antibodies that are easily taken up bythe tumor cells to which they bind. Antibodies of this type find use intherapeutic methods for selective cell killing utilizing antibody-drugor antibody-toxin conjugates ("immunotoxins") wherein a therapeuticantitumor agent is chemically or biologically linked to an antibody orgrowth factor for delivery to the tumor, where the antibody binds to thetumor-associated antigen or receptor with which it is reactive and"delivers" the antitumor agent inside the tumor cells [see, e.g.,Embleton et al., "Antibody Targeting Of Anti-Cancer Agents", inMonoclonal Antibodies For Cancer Detection and Therapy, pp. 317-44(Academic Press, 1985)].

2. Immunotoxins.

Immunotoxins have been investigated as a new approach for treatingmetastatic tumors in man [Pastan and FitzGerald, Science 254:1173-1177(1991); FitzGerald and Pastan, Seminars in Cell Biology 2:31-37 (1991)and Vitetta et al., Science 644:650 (1987)]. Pseudomonas exotoxin A("PE") is a cytotoxic agent produced by Pseudomonas aeruginosa thatkills cells by ADP-ribosylating elongation factor 2, thereby inhibitingprotein synthesis [Iglewski et al., Proc. Natl. Acad. Sci. USA72:2284-2285 (1975)].

PE is a polypeptide comprising three domains [Allured et al., Proc.Natl. Acad. Sci. USA 83:1320-1324 (1986)].

Domain I encodes the cell-binding ability; domain II encodes theproteolytic sensitivity site and the membrane translocation ability; anddomain III encodes the ADP-ribosylation activity of the toxin [Hwang etal., Cell 48:129-136 (1987), Siegall et al., J. Biol. Chem.264:14256-14261 (1989)]. By removing domain I from PE, a truncated 40kDa toxin is formed ("PE40") [Kondo et al., J. Biol. Chem. 263:9470-9475(1988)].

PE40 is weakly toxic to cells because it lacks the cell binding domainfor the PE receptor [Id.] For conjugation of this molecule to anantibody, the amino terminus of PE40 is modified to include a lysineresidue to form "LysPE40" [Batra et al., supra]. Immunotoxins using PE,have shown promise in preclinical models using human tumor xenografts innude mice [Batra et al., Proc. Natl. Acad. Sci. USA 86:8545-8549 (1989);and Pai et al., Proc. Natl. Acad. Sci. USA 88:3358-3362 (1991)].

Several internalizing antibodies reacting with lymphocyte antigens areknown. In contrast, such antibodies are rare when dealing with solidtumors. One of the few examples of an internalizing antibody reactingwith carcinomas is an antibody disclosed in Domingo et al., "TransferrinReceptor As A Target For Antibody-Drug Conjugates," Methods Enzymol.112:238-47 (1985). This antibody is reactive with the humantransferrin-receptor glycoprotein expressed on tumor cells. However,because the transferrin-receptor is also expressed on many normaltissues, and often at high levels, the use of ananti-transferrin-receptor antibody in an antibody-drug or antibody-toxinconjugate may have significant toxic effects on normal cells. Theutility of this antibody for selective killing or inhibition of tumorcells is therefore questionable. Another internalizing antibody is BR64(disclosed in co-pending patent applications U.S. Ser. No. 289,635,filed Dec. 22, 1988, and Ser. No. 443,696 filed Nov. 29, 1989, andincorporated by reference herein), which binds to a large spectrum ofhuman carcinomas.

3. Chimeric Antibodies.

The cell fusion technique for the production of monoclonal antibodies[Kohler and Milstein, Nature (London) 256:495 (1975)] has permitted thedevelopment of a number of murine monoclonal antibodies reactive withantigens, including previously unknown antigens.

However, murine monoclonal antibodies may be recognized as foreignsubstances by the human immune system and neutralized such that theirpotential in human therapy is not realized. Therefore, recent effortshave focused on the production of so-called "chimeric" antibodies by theintroduction of DNA into mammalian cells to obtain expression ofimmunoglobulin genes [Oi et al., Proc. Natl. Acad. Sci. USA 80:825(1983); Potter et al., Proc. Natl. Acad. Sci. USA 81:7161; Morrison etal., Proc. Natl. Acad. Sci. USA 81:6581 (1984); Sahagan et al., J.Immunol. 137:1066 (1986); Sun et al., Proc. Natl. Acad. Sci. 84:214(1987)].

Chimeric antibodies are immunoglobulin molecules comprising a human andnon-human portion. More specifically, the antigen combining region(variable region) of a chimeric antibody is derived from a non-humansource (e.g. murine) and the constant region of the chimeric antibodywhich confers biological effector function to the immunoglobulin isderived from a human source. The chimeric antibody should have theantigen binding specificity of the non-human antibody molecule and theeffector function conferred by the human antibody molecule.

In general, the procedures used to produce chimeric antibodies involvethe following steps:

a) identifying and cloning the correct gene segment encoding the antigenbinding portion of the antibody molecule; this gene segment (known asthe VDJ, variable, diversity and joining regions for heavy chains or VJ,variable, joining regions for light chains or simply as the V orvariable region) may be in either the cDNA or genomic form;

b) cloning the gene segments encoding the constant region or desiredpart thereof;

c) ligating the variable region with the constant region so that thecomplete chimeric antibody is encoded in a form that can be transcribedand translated;

d) ligating this construct into a vector containing a selectable markerand gene control regions such as promoters, enhancers and poly(A)addition signals;

e) amplifying this construct in bacteria;

f) introducing this DNA into eukaryotic cells (transfection) most oftenmammalian lymphocytes;

g) selecting for cells expressing the selectable marker;

h) screening for cells expressing the desired chimeric antibody; and

i) testing the antibody for appropriate binding specificity and effectorfunctions.

Antibodies of several distinct antigen binding specificities have beenmanipulated by these protocols to produce chimeric proteins [e.g.anti-TNP: Boulianne et al., Nature 312:643 (1984); and anti-tumorantigens: Sahagan et al., J. Immunol. 137:1066 (1986)]. Likewise,several different effector functions have been achieved by linking newsequences to those encoding the antigen binding region. Some of theseinclude enzymes [Neuberger et al., Nature 312:604 (1984)],immunoglobulin constant regions from another species and constantregions of another immunoglobulin chain [Sharon et al., Nature 309:364(1984); Tan et al., J. Immunol. 135:3565-3567 (1985)].

4. Modifying Genes in Situ Encoding Monoclonal Antibodies.

The discovery of homologous recombination in mammalian cells permits thetargeting of new sequences to specific chromosomal loci. Homologousrecombination occurs when cultured mammalian cells integrate exogenousDNA into chromosomal DNA at the chromosome location which containssequences homologous to the plasmid sequences [Folger et al., Mol. Cell.Biol. 2:1372-1387 (1982); Folger et al., Symp. Quant. Biol. 49:123-138(1984); Kucherlapati et al., Proc. Natl. Acad. Sci. USA 81:3153-3157(1984); Lin et al., Proc. Natl. Acad. Sci. USA 82:1391-1395 (1985); deSaint Vincent et al., Proc. Natl. Acad. Sci. USA 80:2002-2006 (1983);Shaul et al., Proc. Natl. Acad. Sci. USA 82:3781-3784 (1985)].

The potential for homologous recombination within cells permits themodification of endogenous genes in situ. Conditions have been foundwhere the chromosomal sequence can be modified by introducing into thecell a plasmid DNA which contains a segment of DNA homologous to thetarget locus and a segment of new sequences with the desiredmodification [Thomas et al., Cell 44:419-428 (1986); Smithies et al.,Nature 317:230-234 (1985); Smith et al., Symp. Quant. Biol. 49:171-181(1984)]. Homologous recombination between mammalian cell chromosomal DNAand the exogenous plasmid DNA can result in the integration of theplasmid or in the replacement of some of the chromosomal sequences withhomologous plasmid sequences. This can result in placing a desired newsequence at the endogenous target locus.

The process of homologous recombination has been evaluated using geneswhich offer dominant selection such as NEO and HPRT for a few cell types[Song et al., Proc. Natl. Acad. Sci. USA 84:6820-6824 (1987); Rubinitzand Subramani, Mol. Cell Biol. 6:1608-1614 (1986); and Liskay, Cell35:157-164 (1983)]. Recently, procedures for modifying antibodymolecules and for producing chimeric antibody molecules using homologousrecombination to target gene modification have been described [Fell etal., Proc. Natl. Acad. Sci. USA 86:8507-8511 (1989); and co-pending U.S.patent application Ser. No. 243,873 filed Sep. 14, 1988, and Ser. No.468,035 filed Jan. 22, 1990, assigned to the same assignee as thepresent application, all of which are incorporated by reference herein].

5. Monoclonal Antibodies in Therapy.

The most direct way to apply antitumor monoclonal antibodies clinicallyis to administer them in unmodified form, using monoclonal antibodieswhich display antitumor activity in vitro and in animal (such as humans,dogs, cows, pigs, horses, cats, rats, and mice) models. Most monoclonalantibodies to tumor antigens do not appear to have any antitumoractivity by themselves, but certain monoclonal antibodies are knownwhich mediate complement-dependent cytotoxicity (complement-dependentcytotoxicity), i.e. kill human tumor cells in the presence of humanserum as a source of complement [see, e.g. Hellstrom et al., Proc. Natl.Acad. Sci. USA 82:1499-1502 (1985)], or antibody-dependent cellularcytotoxicity (antibody-dependent cellular cytotoxicity) together witheffector cells such as human NK cells or macrophages.

To detect antibody-dependent cellular cytotoxicity andcomplement-dependent cytotoxicity activity monoclonal antibodies aretested for lysing cultured ⁵¹ Cr-labeled tumor target cells over a4-hour incubation period.

Target cells are labeled with ⁵¹ Cr and then exposed for 4 hours to acombination of effector cells (in the form of human lymphocytes purifiedby the use of a lymphocyte-separation medium) and antibody, which isadded in concentrations varying between 0.1 μg/ml and 10 μg/ml. Therelease of ⁵¹ Cr from the target cells is measured as evidence oftumor-cell lysis (cytotoxicity). Controls include the incubation oftarget cells alone or with either lymphocytes or monoclonal antibodyseparately.

The total amount of ⁵¹ Cr that can be released is measured andantibody-dependent cellular cytotoxicity is calculated as the percentkilling of target cells observed with monoclonal antibody plus effectorcells as compared to target cells being incubated alone. The procedurefor complement-dependent cytotoxicity is identical to the one used todetect antibody-dependent cellular cytotoxicity except that human serum,as a source of complement, (diluted 1:3 to 1:6) is added in place of theeffector cells.

Monoclonal antibodies with antibody-dependent cellular cytotoxicity andcomplement-dependent cytotoxicity activity are considered fortherapeutic use because they often have anti-tumor activities in vivo.Antibodies lacking antibody-dependent cellular cytotoxicity andcomplement-dependent cytotoxicity activity in vitro, on the other hand,are commonly ineffective in vivo unless used as carriers of antitumoragents.

The ability of a monoclonal antibody to activate the host's complementmay prove to be therapeutically beneficial not only because tumor cellsmay be killed, but also because the blood supply to tumors may increase,thus facilitating the uptake of drugs [see Hellstrom et al.,"Immunological Approaches to Tumor Therapy: Monoclonal Antibodies, TumorVaccines, and Anti-Idiotypes, in Covalently Modified Antigens andAntibodies in Diagnosis and Therapy", Quash & Rodwell, eds., MarcelDekker, pp. 15-18 (1989)].

Among mouse monoclonal antibodies, the IgG2a and IgG3 isotypes are mostcommonly associated with antibody-dependent cellular cytotoxicity andcomplement-dependent cytotoxicity. Antibodies having bothantibody-dependent cellular cytotoxicity and complement-dependentcytotoxicity activity have high selectivity for killing only the tumorcells to which they bind and would be unlikely to lead to toxic effectsif non-specifically trapped in lung, liver or other organs. This maygive such antibodies an advantage over radiolabeled antibodies orcertain types of immunoconjugates.

Therapeutic modalities directed to treating tumors are commonlyavailable. For example, chemotherapy is an effective treatment forselected human tumors. However, with chemotherapy only modest progresshas been made for treating the majority of carcinomas, includingcarcinomas of breast, lung, and colon.

The introduction of monoclonal antibody (MAb) technology in the 1970sraised hopes that tumor-specific MAbs could be used to target anti-tumoragents and provide more effective therapy (K. E. Hellstrom, and I.Hellstrom, in: Biologic Therapy of Cancer: Principles and Practice, V.T. DeVita, S. Hellman, and S. A. Rosenberg, Eds. (J. P. LippincottCompany, Philadelphia, Pa., 1991) pp. 35-52).

6. Immunoconjugates in Therapy.

Various immunoconjugates in which antibodies were used to targetchemotherapeutic drugs (P. N. Kularni, A. H. Blair, T. I. Ghose, CancerRes. 41, 2700 (1981); R. Arnon, R. and M. Sela, Immunol. Rev. 62, 5(1982); H. M. Yang and R. A. Resifeld, Proc. Natl. Acad. Sci. U.S.A.,85, 1189 (1988); R. O. Dilman, D. E. Johnson, D. L. Shawler, J. A.Koziol, Cancer Res. 48, 6097 (1988); L. B. Shih, R. M. Sharkey, F. J.Primus, D. M. Goldenberg, Int. J. Cancer 41, 832 (1988); P. A. Trail, etal., Cancer Res. 52, 5693 (1992)), or plant and bacterial toxins (I.Pastan, M. C. Willingham, D. J. Fitzgerald, Cell 47, 641 (1986); D. C.Blakey, E. J. Wawrzynczak, P. M. Wallace, P. E. Thorpe, in MonoclonalAntibody Therapy Prog. Allergy, H. Waldmann, Ed. (Karger, Basel, 1988),pp. 50-90) have been evaluated in preclinical models and found to beactive in vitro and in vivo.

However, activity of these MAbs was usually assessed against newlyimplanted rather than established tumors and was typically superior tomatching, but not optimal, doses of the unconjugated drug.

Although conjugates have been described with anti-tumor activity againstestablished tumors that were superior to that of an optimal dose ofunconjugated drug, the therapeutic index was low and superior activitywas achieved only at or near the maximum tolerated dose (MTD) of theconjugate (P. A. Trail, et al., Cancer Res. 52, 5693 (1992)).

The results of clinical studies of drug and toxin conjugates (i.e.,immunoconjugates) have also been disappointing, particularly for solidtumors (E. S. Vitetta, R. J. Fulton, R. D. May, M. Till, J. W. Uhr,Science 238, 1098 (1987); H. G. Eichler, Biotherapy 3, 11 (1991); E.Wawrzynczak, Br. J. Cancer 64, 624 (1991); G. A. Pietersz and I. F. C.McKenzie, Immunol. Rev. 129, 57 (1992)).

Very few antibodies are able to kill tumor cells by themselves, that is,in the absence of effector cells or complement as in antibody-dependentcellular cytotoxicity or complement-dependent cytotoxicity. BR96 is suchan antibody, because it can kill cells by itself at an antibodyconcentration of approximately 10 μg/ml or higher. Such antibodies areof particular interest since they can interfere with some key event inthe survival of neoplastic cells.

Presently, chemotherapeutic agents, by themselves, do not distinguishbetween malignant and normal cells. They are absorbed by both celltypes. Tumors that are detected early on such as acute lymphocyticleukemia and lymphomas are highly susceptible to drugs.

Tumors that are hidden until growth has reached a plateau, such ascancer of the lung and colon, have little sensitivity to drugs. Normalcells with high growth fraction are inevitably attacked by today'santi-cancer drugs, explaining the prevalence of severe side effects inthe gastrointestinal tract and of hair loss. This holds true whether thecytotoxicity of the drug is due to alkylation, intercalation, ordisruption of biosynthesis/antimetabolites.

The molecules of the invention, e.g., the immunotoxins, are homogeneousmolecules that retain the specificity of the cell binding portion withthe cytotoxic potential of the toxin.

It is thus apparent that antibodies, antibody conjugates andimmunotoxins that display a high degree of selectivity to a wide rangeof carcinomas, have anti-tumor activity, and are capable of beingreadily internalized by tumor cells, may be of great benefit in tumortherapy.

SUMMARY OF THE INVENTION

The present invention provides internalizing antibodies, antibodyconjugates and recombinant, single-chain immunotoxins that are highlyselective for a range of human carcinomas. More specifically, the novelantibodies of the invention, designated as BR96 antibodies, are a murinemonoclonal antibody and a chimeric antibody that bind to a cell membraneantigen found on human carcinoma cells.

The novel conjugates and single-chain immunotoxins contain an exotoxinsuch as PE and bind to the antigen on tumor cells. The antibodies,conjugates and single-chain immunotoxins are highly reactive withcarcinoma cells, such as those derived from breast, lung, colon andovarian carcinomas, showing no or limited reactivity with normal humancells or other types of tumors such as lymphomas or sarcomas. Inaddition, the antibodies of the invention internalize within thecarcinoma cells to which they bind and they are capable of killing tumorcells by themselves, i.e., not in conjugated form, and without effectorcells or complement.

Thus the BR96 antibodies are of particular use in therapeuticapplications, for example to react with tumor cells, and in conjugatesand single-chain immunotoxins as target-selective carriers of variousagents which have antitumor effects including chemotherapeutic drugs,toxins, immunological response modifiers, enzymes and radioisotopes. Theantibodies can thus be used as a component of various immunoconjugatesincluding antibody-drug and antibody-toxin conjugates, including ricinand PE-antibodies and ricin and PE-antibody fragment immunotoxins, whereinternalization of the conjugate is favored, and after radiolabeling todeliver radioisotope to tumors. The BR96 antibodies can also betherapeutically beneficial even in the unmodified form. Furthermore, theantibodies are useful for in vitro or in vivo diagnostic methodsdesigned to detect carcinomas.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the percent inhibition of thymidine incorporation intothe DNA of 3396 breast carcinoma cells treated with a BR96-RAimmunotoxin at varying concentrations as described in Example 3, infra.BR6-RA is an internalizing antibody which is used as a negative controlbecause it does not bind to the 3396 cells.

FIG. 2 depicts the percent inhibition of thymidine incorporation intothe DNA of 2707 lung carcinoma cells treated with a BR96-PA immunotoxinat varying concentrations as described in Example 3, infra. BR6-RA is aninternalizing antibody which also binds to the 2707 cells.

FIG. 3 depicts the percent inhibition of thymidine incorporation intothe DNA of HCT116 colon carcinoma cells treated with a BR96-RAimmunotoxin at varying concentrations as described in Example 3, infra.BR96 does not bind to HCT 116 cells.

FIG. 4 depicts the percent inhibition of thymidine incorporation intothe DNA of C colon carcinoma cells treated with a BR96-RA immunotoxin atvarying concentrations as described in Example 3, infra. BR6-RA does notbind to the C cells; L6-RA binds to the C cells but does notinternalize.

FIG. 5 depicts the percent inhibition of thymidine incorporation intothe DNA of 3347 colon carcinoma cells treated with a BR96-RA immunotoxinat varying concentrations as described in Example 3, infra. BR96 doesnot bind to these cells while BR6 does.

FIG. 6 depicts the results of FACS analysis of the cytotoxicity ofpropidium iodide stained 3396 breast carcinoma cells, 2987 lungcarcinoma cells and 3619 colon carcinoma cells, as described in Example4, infra.

FIG. 7 depicts the effects of BR96 on cell proliferation of various celllines as described in Example 4, infra.

FIG. 8 illustrates the effect of BR96 on cell growth of various celllines, measured by a staining method as described in Example 4, infra.

FIG. 9 illustrates the results of tests to determine antibody-dependentcellular cytotoxicity activity of BR96 as described in Example 5, infra.

FIG. 10 describes the results of tests to determine complement-dependentcytotoxicity activity of BR96 as described in Example 6, infra.

FIG. 11 is a bar graph of the results of testing the reactivity of BR96against glycolipids as described in Example 7, infra.

FIG. 12 is a bar graph of the results of testing the reactivity of BR96against neoglycoproteins as described in Example 7, infra.

FIG. 13 is a graph of the binding activity of BR96 F(ab')₂ fragmentscompared to that of whole BR96 monoclonal antibody in an ELISA usinggoat anti-K light chain detecting reagent, as described in Example 8,infra.

FIG. 14 is a graph of the binding activity of BR96 F(ab')₂ fragments ascompared to that of whole BR96 monoclonal antibody in an ELISA usingperoxidase conjugated protein A detecting reagent, as described inExample 8, infra.

FIG. 15 is a diagram of vector phγ₁ HC-D used in the electroporationprocedure, as described in Example 9, infra.

FIG. 16 is a diagram of vector pSV₂ gpt/C_(K) used in theelectroporation procedure, as described in Example 9, infra.

FIG. 17 is a graph depicting the results of the competition bindingassay comparing the binding of the murine BR96 monoclonal antibody ofthe invention with binding of the chimeric BR96 antibody of theinvention, as described in Example 9, infra.

FIG. 18 depicts the results of FACS analysis of the cytotoxicity of theantibodies of the invention on 3396 breast carcinoma cells as describedin Example 10, infra.

FIG. 19 depicts the results of FACS analysis of the cytotoxicity of theantibodies of the invention on 2987 human lung adenocarcinoma cells asdescribed in Example 10, infra.

FIG. 20 depicts the results of FACS analysis of the cytotoxicity of theantibodies of the invention on MCF-7 cells as described in Example 10,infra.

FIG. 21 depicts the percent inhibition of thymidine incorporation intothe DNA of 3396 breast carcinoma cells treated with a murine BR96-RAimmunotoxin and chimeric (Chi)BR96-RA at varying concentrations asdescribed in Example 10, infra.

FIG. 22 depicts the percent inhibition of thymidine incorporation intothe DNA of 3630 breast carcinoma cells treated with a murine BR96-RAimmunotoxin and ChiBR96-RA at varying concentrations, as described inExample 10, infra.

FIG. 23 is a graph depicting the antitumor effects of unmodified BR96 onthe tumor cell line H2987, as described in Example 11, infra.

FIG. 24 is a bar graph illustrating the absence of tumors at the end oftreatment for animals treated with BR96, as described in Example 11,infra.

FIG. 25 depicts the dose effects of BR96 antibody after implantation ofH2707 cells, as determined by tumor volume, as described in Example 11,infra.

FIG. 26 illustrates the effects of treatment with F(ab')₂ fragments andchimeric BR96 after implantation of 2707 cells as determined by tumorvolume, as described in Example 11, infra.

FIG. 27 illustrates the absence of tumors after treatment with variousdoses of BR96 antibody, as compared to the effects of F(ab')₂ fragmentsand chimeric BR96, as described in Example 11, infra.

FIG. 28 is a photograph of the gel obtained from non-reducing SDS-PAGEanalysis of conjugated and unconjugated ChiBR96 IgG, Fab' and F(ab')₂immunotoxins as described in Example 13, infra (Lane 1: ChiBR96 IgG;Lane 2: ChiBR96 Fab'; Lane 3: ChiBR96 Fab'-LysPE40; Lane 4: Native PE;Lane 5: LysPE40; Lane 6:ChiBR96 IgG-LysPE40; Lane 7:ChiBR96(Fab')₂ ;Lane 8: ChiBR96 F(ab')₂ -LysPE40; Lane 9:ChiBR96 IgG-LysPE40; Lane10:ChiBR96 IgG).

FIG. 29 is a graph depicting the results of competition of ChiBR96-PEand ChiBR96-LysPE40 binding as described in Example 13, infra (ChiBR96(closed circle); ChiBR96-PE (closed square); ChiBR96-LysPE40 (opentriangle)).

FIGS. 30A, B, C are graphs of the direct binding of intactChiBR96-LysPE40, F(ab')₂ -LysPE40 and Fab'-LysPE40 to L2987 cells, asdescribed in Example 13, infra (ChiBR96 (closed circle); ChiBR96-LysPE40(open triangle); ChiBR96 F(ab')₂ (open square); ChiBR96 F(ab')₂ -LysPE40(closed circle); ChiBR96 Fab' (open circle); ChiBR96 Fab'-LysPE40 (opencircle)).

FIGS. 31A and B are graphs showing the determination of endocytosis ofcell-surface immunotoxin after modulation with ChiBR96-PE orChiBR96-LysPE40 immunotoxins as described in Example 13, infra (31A:loss of cell surface immunotoxin under modulating and non-modulatingconditions; 31B: internalization of cell-bound immunotoxin usingimmunotoxin plus radiolabeled M-40/1 complex; ChiBR96-PE coated cellswere incubated at 4° C. (open circle) or 37° C. (closed circle);ChiBR96-LysPE40 coated cells were incubated at 4° C. (open triangle) or37° C. (closed triangle).

FIG. 32 is a graph of the cytotoxic effects of various ChiBR96 formsconjugated to LysPE40 against MCF-7 cells as described in Example 13,infra (ChiBR96-PE40 (closed square); ChiBR96 F(ab')₂ -PE40 (closedcircle); ChiBR96 Fab'-PE40 (closed triangle); PE40 open circle).

FIG. 33 is a bar graph depicting the results of competition analysis ofChiBR96-PE40 cytotoxic activity against MCF-7 cells as described inExample 13, infra.

FIGS. 34A and B are graphs showing the results of protein synthesisinhibition analysis of ChiBR96-(PE/LysPE40) vs. PE against MCF-7 cellsas described in Example 13, infra (34A: 1 hr; 34B: 20hr; ChiBR96-PE(closed square); BR96-PE40 (closed circle); PE (closed triangle).

FIG. 35 (SEQ ID NO: 3) is the DNA and amino acid sequence for BR96 sFvencoded by plasmid pBR96 Fv, as described in Example 14, infra.

FIG. 36 is a schematic illustration of the construction of expressionplasmid pBW 7.0 encoding BR96 sFv-PE40 as described in Example 14, infra(E, Eco RI; H, Hind III; K, KPNI; N, NDe I; S, Sal I; (Gly₄ Ser)₃represents a 15 amino acid linker).

FIGS. 37A, B, C illustrate the purification of BR96 sFv-PE40 by gelfiltration as described in Example 14, infra (FIG. 37A: profile of gelfiltration column chromatography of renatured BR96 sFv-PE40 afterinitial purification over Q-Sepharose; FIG. 37B: 12% denaturingSDS-polyacrylamide gel stained with Coomassie brilliant blue; FIG. 37C:immunoblot of a 4-12% non-denaturing SDS-polyacrylamide gel probed withBR96 anti-idiotypic antibody; lanes 1-15 correspond to fractions 7-21 onthe gel filtration profile shown in FIG. 37A; lane M representsmolecular weight marker proteins in kilodaltons. Molecular weightstandards correspond to 670 kDa, 158 kDa, 44 kDa and 17 kDa eluted infractions 10, 15, 21 and 30, respectively).

FIG. 38 is a graph depicting the results of a direct binding assay onELISA plates coated with Lewis-Y antigen and probed with BR96anti-idiotype antibody, and comparing the binding of BR96 IgG (opensquare), BR96 sFv-PE40 monomers (closed circle), BR96 sFv-PE40aggregates (closed triangle) and L6 IgG (open circle), as described inExample 14, infra.

FIG. 39 is a graph showing the results of binding analysis of BR96sFv-PE40, with competition of ¹²⁵ I-labeled BR96 IgG with BR96 sFv-PE40(closed circle), BR96 IgG (open square) and L6 IgG (open circle), asdescribed in Example 14, infra.

FIG. 40 is a graph showing the results of cytotoxicity analysis of BR96sFv-PE40 inhibition of protein synthesis in MCF-7 cells as described inExample 14, infra (BR96 sFv-PE40 (closed circle) and ChiBR96-LysPE40(closed square)).

FIG. 41 are histograms of FACS analysis of five human carcinoma lines asdescribed in Example 14, infra (data is displayed in each histogram asthe mean channel number for BR96 IgG or a human IgG control antibody.Fluorescence intensity for each cell line is determined by subtractingthe human IgG mean channel number from the BR96 mean channel number).

FIG. 42 is a bar graph showing the results of competitive cytotoxicanalysis of BR96 sFv-PE40 inhibition of protein synthesis in L2987 cellsby BR96 sFv-PE40 (50 ng/ml) alone or in the presence of either BR96 IgGor L6 IgG (100 μg/ml) as described in Example 14, infra.

FIG. 43 is a graph showing anti-tumor effects of BR96-sFvPE40 in vivoagainst MCF-7 human breast tumor xenografts, as described in Example 15,infra (ADM 6 mg/kg (closed square), BR96-sFvPE40 0.50 mg/kg (closedcircle), BR96-sFvPE40 0.75 mg/kg (open circle), control (closedtriangle)).

FIG. 44 is a graph showing anti-tumor activity of BR96-immunotoxinsagainst L2987 human lung tumor xenografts as described in Example 15,infra (sFv-PE40 0.125 mg/kg (closed triangle), sFv-PE40 0.25 mg/kg(closed circle), sFv-PE40 0.375 mg/kg (open circle), IgG Lys-PE40conjugate 1.25 mg/kg (closed square), Interleukin 6-PE40 0.375 mg/kg(open square), control (closed triangle)).

FIG. 45 is a drawing of the structure of BR96-DOX.

FIGS. 46A-D are line graphs showing the antigen-specific antitumoractivity of BR96-DOX.

(A) Control animals (closed square); animals treated with BR96-DOX (5mg/kg DOX) (closed circle), IgG-DOX (5 mg/kg DOX) (closed triangle); oroptimized DOX (8 mg/kg) (open square).

(B) Control animals (closed square); animals treated with BR96-DOX (10mg/kg) (closed circle), IgG-DOX (10 mg/kg) (closed triangle), oroptimized DOX (8 mg/kg) (open square).

(C) Control animals (closed square); animals treated with BR96-DOX (5mg/kg) (closed circle), IgG-DOX (5 mg/kg) (closed triangle), or DOX (6mg/kg) (open square).

(D) Control animals (closed square) animals treated with BR96-DOX (8mg/kg) (closed circle), or DOX (8 mg/kg) (open triangle).

FIG. 47 is a line graph showing that BR96-DOX cures athymic mice oflarge disseminated tumors. Untreated controls (closed triangle),BR96-DOX treated (8 mg/kg) (closed circle) or DOX treated (8 mg/kg)(closed square) 82, 86 and 90 days after inoculation of tumor cells.

FIG. 48 is a line graph showing that BR96-DOX cures human lung tumorsimplanted in athymic rats. Control animals (closed square), animalstreated with BR96-DOX (4 mg/kg) (open circle), BR96-DOX (2 mg/kg)(closed triangle), or DOX (4 mg/kg) (open square).

FIG. 49(a) provides a synthetic scheme for preparing a thiolatedantibody using SPDP as the thiolation agent.

FIG. 49(b) provides a synthetic scheme for preparing an immunoconjugateof the invention in which the ligand is a SPDP-thiolated antibody.

FIG. 49(c) provides a synthetic scheme for preparing an immunoconjugateof the invention in which the ligand is an iminothiolane-thiolatedantibody.

FIG. 50 shows a process for reducing with DTT an antibody to prepare a"relaxed" antibody and synthesis of an immunoconjugate of the invention.

FIG. 51 provides in vitro cytotoxic activity data for BR64-Adriamycinconjugates of the invention against L2987 tumors.

FIG. 52 provides in vivo cytotoxic activity data for BR64-Adriamycinconjugates of the invention against L2987 tumors.

FIGS. 53A/B/C provides comparative in vivo cytotoxic data forcombination therapy using BR64, Adriamycin and non-binding conjugate(SN7-Adriamycin).

FIG. 54 provides in vivo cytotoxic activity data for Bombesin-Adriamycinconjugates of the invention against H345 tumors.

FIG. 55 provides in vitro cytotoxic activity data for Adriamycinconjugates of relaxed chimeric BR96 and SPDP-thiolated chimeric BR96.

FIG. 56 provides in vivo cytotoxic activity data for Adriamycinconjugates of relaxed BR64 and relaxed chimeric L6 against L2987 tumors.

FIGS. 57 to 59 provide in vivo cytotoxic activity data against L2987tumors for Adriamycin conjugates of relaxed chimeric BR96 compared tofree Adriamycin and non-binding conjugates.

FIG. 60 provides in vivo cytotoxic activity data for Adriamycinconjugates of relaxed chimeric BR96 against RCA Human Breast Tumors.

FIG. 61 provides in vivo cytotoxic activity data for Adriamycinconjugates of relaxed chimeric BR96 against RCA Human Colon Tumors.

FIG. 62 provides a graph of the effect on --SH titer as a function ofmole ratio of DTT to antibody in the preparation, under an inertatmosphere, of a relaxed antibody.

DETAILED DESCRIPTION OF THE INVENTION

DEFINITIONS

As used in this application, the following words or phrases have themeanings specified.

As used herein, "functional equivalent" means being capable of (1)binding the antigen binding site to which BR96 is directed (i.e.competitively inhibit the antigen binding site), (2) binding carcinomacells, (3) being internalized within the carcinoma cells to which theybind, and/or (4) mediating ADCC and CDC effector functions.

As used herein, "joined" means to couple directly or indirectly onemolecule with another by whatever means, e.g., by covalent bonding, bynon-covalent bonding, by ionic bonding, or by non-ionic bonding.Covalent bonding includes bonding by various linkers such as thioetherlinkers or thioester linkers. Direct coupling involves one moleculeattached to the molecule of interest. Indirect coupling involves onemolecule attached to another molecule not of interest which in turn isattached directly or indirectly to the molecule of interest.

As used herein, "recombinant molecule" means a molecule produced bygenetic engineering methods.

As used herein, "fragment" is defined as at least a portion of thevariable region of the immunoglobulin molecule which binds to itstarget, i.e. the antigen binding region. Some of the constant region ofthe immunoglobulin may be included.

As used herein, an "immunoconjugate" means any molecule or ligand suchas an antibody or growth factor chemically or biologically linked to acytotoxin, a radioactive agent, an anti-tumor drug or a therapeuticagent. The antibody or growth factor may be linked to the cytotoxin,radioactive agent, anti-tumor drug or therapeutic agent at any locationalong the molecule so long as it is able to bind its target. Examples ofimmunoconjugates include immunotoxins and antibody conjugates.

As used herein, "selectively killing" means killing those cells to whichthe antibody binds.

As used herein, examples of "carcinomas" include bladder, breast, colon,liver, lung, ovarian, and pancreatic carcinomas.

As used herein, "immunotoxin" means an antibody or growth factorchemically or biologically linked to a cytotoxin or cytotoxic agent.

As used herein, an "effective amount" is an amount of the antibody,immunoconjugate, recombinant molecule which kills cells or inhibits theproliferation thereof.

As used herein, "competitively inhibits" means being capable of bindingto the same target as another molecule. With regard to an antibody,competitively inhibits mean that the antibody is capable of recognizingand binding the same antigen binding region to which another antibody isdirected.

As used herein, "antigen-binding region" means that part of theantibody, recombinant molecule, the fusion protein, or theimmunoconjugate of the invention which recognizes the target or portionsthereof.

As used herein, "therapeutic agent" means any agent useful for therapyincluding anti-tumor drugs, cytotoxins, cytotoxin agents, andradioactive agents.

As used herein, "anti-tumor drug" means any agent useful to combatcancer including, but not limited to, cytotoxins and agents such asantimetabolites, alkylating agents, anthracyclines, antibiotics,antimitotic agents, procarbazine, hydroxyurea, asparaginase,corticosteroids, mytotane (O,P'-(DDD)), interferons and radioactiveagents.

As used herein, "a cytotoxin or cytotoxic agent" means any agent that isdetrimental to cells. Examples include taxol, cytochalasin B, gramicidinD, ethidium bromide, emetine, mitomycin, etoposide, tenoposide,vincristine, vinblastine, colchicin, doxorubicin, daunorubicin,dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D,1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,propranolol, and puromycin and analogs or homologs thereof.

As used herein, "a radioactive agent" includes any radioisotope which iseffective in destroying a tumor. Examples include, but are not limitedto, cobalt-60 and X-rays. Additionally, naturally occurring radioactiveelements such as uranium, radium, and thorium which typically representmixtures of radioisotopes, are suitable examples of a radioactive agent.

As used herein, "administering" means oral administration,administration as a suppository, topical contact, intravenous,intraperitoneal, intramuscular or subcutaneous administration, or theimplantation of a slow-release device such as a miniosmotic pump, to thesubject.

As used herein, "directly" means the use of antibodies coupled to alabel. The specimen is incubated with the labeled antibody, unboundantibody is removed by washing, and the specimen may be examined.

As used herein, "indirectly" means incubating the specimen with anunconjugated antibody, washing and incubating with afluorochrome-conjugated antibody. The second or "sandwich" antibody thusreveals the presence of the first.

As used herein "reacting" means to recognize and bind the target. Thebinding may be non-specific. Specific binding is preferred.

As used herein, "curing" means to provide substantially complete tumorregression so that the tumor is not palpable for a period of time, i.e.,≧10 tumor volume doubling delays (TVDD=the time in days that it takesfor control tumors to double in size).

As used herein, "tumor associated antigens" means any cell surfaceantigen which is generally associated with tumor cells, i.e., occurringto a greater extent as compared with normal cells. Such antigens may betumor specific. Alternatively, such antigens may be found on the cellsurface of both tumorigenic and non-tumorigenic cells. These antigensneed not be tumor specific. However, they are generally more frequentlyassociated with tumor cells than they are associated with normal cells.

As used herein, "tumor targeted antibody" means any antibody whichrecognizes cell surface antigens on tumor (i.e., cancer) cells. Althoughsuch antibodies need not be tumor specific, they are tumor selective,i.e. bind tumor cells more so than it does normal cells.

As used herein, "internalizing tumor targeted antibody" includes anytumor targeted antibody which is easily taken up by the tumor cells towhich they bind.

As used herein, "internalizing tumor targeted antibody which recognizesthe Le^(y) determinant" includes internalizing tumor targeted antibodywhich specifically recognizes at least a portion of the Le^(y)determinant.

As used herein, "inhibit proliferation" means to interfere with cellgrowth by whatever means.

As used herein, "mammalian tumor cells" include cells from animals suchas human, ovine, porcine, murine, bovine animals.

As used herein, "pharmaceutically acceptable carrier" includes anymaterial which when combined with the antibody retains the antibody'simmunogenicity and non-reactive with the subject's immune systems.Examples include, but are not limited to, any of the standardpharmaceutical carriers such as a phosphate buffered saline solution,water, emulsions such as oil/water emulsion, and various types ofwetting agents. Other carriers may also include sterile solutions,tablets including coated tablets and capsules.

Typically such carriers contain excipients such as starch, milk, sugar,certain types of clay, gelatin, stearic acid or salts thereof, magnesiumor calcium stearate, talc, vegetable fats or oils, gums, glycols, orother known excipients. Such carriers may also include flavor and coloradditives or other ingredients. Compositions comprising such carriersare formulated by well known conventional methods.

In order that the invention herein described may be more fullyunderstood, the following description is set forth.

1. Novel Antibodies of the Invention.

The present invention relates to novel antibodies that are highlyspecific for carcinoma cells. More particularly, the antibodies reactwith a range of carcinomas such as breast, lung, ovary and coloncarcinomas, while showing none or limited reactivity with normal humantissues or other types of tumors such as sarcomas or lymphomas.

One type of novel antibodies of the invention is designated BR96. TheBR96 antibodies can be used to isolate and characterize the antigen towhich they bind. Thus, the BR96 antibodies can be used as a probe toidentify and characterize the epitope recognized and to further definethe cell membrane antigen with which they react [see, e.g., Nudelman etal., "Characterization of Human Melanoma-Associated Ganglioside AntigenDefined By A Monoclonal Antibody, 4.2", J. Biol. Chem., 257 (1)12752-56(1982) and Hakomori, "Tumor Associated Carbohydrate Antigens", Ann. Rev.Immunol., 2:103-26 (1984)].

BR96 recognizes as at least part of its binding site a portion of anepitope of a Le^(y) carbohydrate determinant which is a portion of anantigen abundantly expressed on carcinomas of the colon, breast, ovary,and lung and, to a lesser extent, on epithelial cells from thegastrointestinal tract. Further, BR96 in the absence of effector cellsor complement can inhibit tumor cell DNA synthesis.

Results of preliminary epitope screens conducted on monoclonal antibodyBR96 have indicated that the epitope which is a portion of the antigenon the carcinoma cells to which BR96 antibody binds is a fucosylatedvariant of Lewis Y (Le^(y)). Le^(y) has been described by Abe et al., J.Biol. Chem. 258:8934 (1983); Lloyd et al., Immunogenetics 17:537 (1983);Brown et al., Biosci. Rep. 3:163 (1983); and Hellstrom et al., CancerRes. 46:3917 (1986). Certain fucosylated variants of Lewis Y have beendescribed by Abe et al., Cancer Res. 46:2639-2644 (1986).

The monoclonal antibody of the invention can be produced usingwell-established hybridoma techniques first introduced by Kohler andMilstein [see, Kohler and Milstein, "Continuous Cultures Of Fused CellsSecreting Antibody Of Pre-Defined Specificity", Nature, 256:495-97(1975). See, also, Brown et al., "Structural Characterization Of HumanMelanoma-Associated Antigen p97 with Monoclonal Antibodies", J.Immunol., 127 (2):539-46 (1981)]; Brown et al., "Protein Antigens OfNormal And Malignant Human Cells Identified By Immunoprecipitation WithMonoclonal Antibodies", J. Biol. Chem., 255:4980-83 (1980); Yeh et al.,"Cell Surface Antigens Of Human Melanoma Identified By MonoclonalAntibody", Proc. Natl. Acad. Sci. USA, 76(6):297-31 (1979); and Yeh etal., "A Cell-Surface Antigen Which is Present In the GangliosideFraction And Shared By Human Melanomas", Int. J. Cancer, 29:269-75(1982)].

These techniques involve the injection of an immunogen (e.g., cells orcellular extracts carrying the antigen or purified antigen) into ananimal (e.g., a mouse) so as to elicit a desired immune response (i.e.,antibodies) in that animal. After a sufficient time, antibody-producinglymphocytes are obtained from the animal either from the spleen, lymphnodes or peripheral blood. Preferably, the lymphocytes are obtained fromthe spleen. The splenic lymphocytes are then fused with a myeloma cellline, usually in the presence of a fusing agent such as polyethyleneglycol (PEG). Any of a number of myeloma cell lines may be used as afusion partner according to standard techniques; for example, theP3-NS1/1Ag4-1, P3-x63-Ag8.653 or Sp2/O Ag14 myeloma lines. These myelomalines are available from the American Type Culture Collection ("ATCC")in Rockville, Md.

The resulting cells, which include the desired hybridomas, are thengrown in a selective medium, such as HAT medium, in which unfusedparental myeloma or lymphocyte cells eventually die. Only the hybridomacells survive and can be grown under limiting conditions to obtainisolated clones. The supernatants of the hybridomas are screened for thepresence of antibody of that desired specificity, e.g., by immunoassaytechniques using the antigen that had been used for immunization.Positive clones can then be subcloned under limiting dilution conditionsand the monoclonal antibody produced can be isolated. Hybridomasproduced according to these methods can be propagated in vitro or invivo (in ascites fluid) using techniques known in the art [see,generally, Fink et al., supra at page 123, FIGS. 6-11]. Commonly usedmethods for purifying monoclonal antibodies include ammonium sulfateprecipitation, ion exchange chromatography, and affinity chromatography[see, e.g., Zola et al., "Techniques For The Production AndCharacterization Of Monoclonal Hybridoma Antibodies", in MonoclonalHybridoma Antibodies: Techniques And Applications, Hurell (ed.), pp.51-52 (CRC Press 1982)].

According to a preferred embodiment, a monoclonal antibody of thisinvention, designated BR96, was produced via the hybridoma techniquesdescribed hereinbelow using a breast cancer cell line 3396 as theimmunogen. The BR96 hybridoma, prepared as described hereinbelow andproducing the BR96 antibody, was deposited on Feb. 22, 1989 with theAmerican Type Culture Collection (ATCC), 12301 Parklawn Drive,Rockville, Md. 20852 and has there been identified as follows:

BR96 ATCC Accession No.: HB 10036

The BR96 antibody is of the IgG3 subclass. The antibody displays a highspecificity for carcinoma cells of different organ types, for example,tumors of the breast, lung, colon and ovary as well as cultured celllines established from various breast, lung and colon carcinomas.Furthermore, the BR96 antibody shows no binding to other types of tumorcells such as the T-cell lymphoma cells lines, CEM and MOLT-4, the Bcell lymphoma cell line P3HR-1 or melanoma cell lines. The BR96 antibodyis able to be internalized in antigen-positive tumor cells, is toxic toantigen-positive tumor cells, mediates antibody-dependent cellularcytotoxicity and complement-dependent cytotoxicity activity, andsurprisingly, is cytotoxic alone, i.e. in unmodified form. The BR96antibodies appear to recognize a novel epitope of the Le^(y)determinant.

The present invention provides an immunoconjugate comprising a moleculehaving the antigen-binding region of the BR96 monoclonal antibody joinedto doxorubicin. It would be clear that doxorubicin may be joined at anylocation along the molecule so long as it retains its ability to bindits target. Doxorubicin may be joined by any means including chemicaland biological means.

Clearly analogs and homologs of doxorubicin are encompassed by theinvention. For example, an improved analog of doxorubicin is Fe-chelate.

2. Fragments of the Monoclonal Antibodies of the Invention.

According to another embodiment, F(ab')₂ fragments of the BR96monoclonal antibody were produced by pepsin digestion of purified BR96[Lamoyi, "Preparation of F(ab')₂ Fragments from Mouse IgG of VariousSubclasses", Methods of Enzymol. 121:652-663 (1986)], as describedhereinbelow. The binding of the F(ab')₂ fragments to tumor (3396) andMCF7 cells was shown to be comparable to the binding of the whole BR96monoclonal antibody.

3. Chimeric Antibodies of the Invention.

In another preferred embodiment, a chimeric (murine/human) antibody ofthe invention was produced using a two-step homologous recombinationprocedure as described by Fell et al., in Proc. Natl. Acad. Sci. USA86:8507-8511 (1989) and in co-pending patent application U.S. Ser. No.243,873, filed Sep. 14, 1988, and Ser. No. 468,035, filed Jun. 22, 1990,assigned to the same assignee as the present application; thedisclosures of all of these documents are incorporated in their entiretyby reference herein. This two-step protocol involves use of a targetvector encoding human IgGγ1 heavy chain to transfect a mouse hybridomacell line expressing murine BR96 monoclonal antibody (hybridoma ATCC No.HB 10036) to produce a hybridoma expressing a BR96 chimeric antibodycontaining human IgGγ1 heavy chain. This hybridoma is then transfectedwith a target vector containing DNA encoding human kappa (K) light chainto produce a murine hybridoma expressing a BR96 chimeric antibodycontaining human IgGγ1 heavy chain and human K light chain. The targetvectors used to transfect the hybridomas are the phγ1HC-D vectordigested with Xba1 enzyme (Bristol-Myers Squibb Co., Seattle, Wash.,NRRL No. B 18599) and the HindIII digested pSV₂ gpt/C_(K) vector(Bristol-Myers Squibb Co., Seattle, Wash., NRRL No. B 18507).

The chimeric BR96 hybridoma, identified herein as ChiBR96, prepared asdescribed hereinbelow and producing the chimeric human/murine BR96antibody, was deposited on May 23, 1990, with the ATCC, 12301 ParklawnDrive, Rockville, Md. 20852 and has there been identified as follows:

ChiBR96 ATCC Accession No.: HB 10460

Once the hybridoma that expresses the chimeric antibody is identified,the hybridoma is cultured and a desired chimeric molecules are isolatedfrom the cell culture supernatant using techniques well known in the artfor isolating monoclonal antibodies.

The term "BR96 antibody" as used herein includes whole, intactpolyclonal and monoclonal antibody materials such as the murine BR96monoclonal antibody produced by hybridoma ATCC No. HB 10036, andchimeric antibody molecules such as chimeric BR96 antibody produced byhybridoma ATCC No. 10460. The BR96 antibody described above includes anyfragments thereof containing the active antigen-binding region of theantibody such as Fab, F(ab')₂ and Fv fragments, using techniques wellestablished in the art [see, e.g., Rousseaux et al., "Optimal ConditionsFor The Preparation of Proteolytic Fragments From Monoclonal IgG ofDifferent Rat IgG Subclasses", in Methods Enzymol., 121:663-69 (AcademicPress 1986)]. The BR96 antibody of the invention also includes fusionproteins.

In addition, the BR96 antibody of this invention does not display anyimmunohistologically detectable binding to normal human tissues frommajor organs, such as kidney, spleen, liver, skin, lung, breast, colon,brain, thyroid, heart, lymph nodes or ovary. Nor does the antibody reactwith peripheral blood leukocytes. BR96 antibody displays limited bindingto some cells in the tonsils and testes, and binds to acinar cells inthe pancreas, and to epithelial cells in the stomach and esophagus.Thus, the BR96 antibody is superior to most known antitumor antibodiesin the high degree of specificity for tumor cells as compared to normalcells [see, e.g., Hellstrom et al., "Immunological Approaches To TumorTherapy: Monoclonal Antibodies, Tumor Vaccines, And Anti-Idiotypes", inCovalently Modified Antigens And Antibodies In Diagnosis And Therapy,Quash/Rodwell (eds.), pp. 1-39 (Marcel Dekker, Inc., 1989) and Bagshawe,"Tumour Markers--Where Do We Go From Here", Br. J. Cancer, 48:167-75(1983)].

Also included within the scope of the invention are anti-idiotypicantibodies to the BR96 antibody of the invention. These anti-idiotypicantibodies can be produced using the BR96 antibody and/or the fragmentsthereof as immunogen and are useful for diagnostic purposes in detectinghumoral response to tumors and in therapeutic applications, e.g., in avaccine, to induce an anti-tumor response in patients [see, e.g., Nepomet al., "Anti-Idiotypic Antibodies And The Induction Of Specific TumorImmunity", in Cancer And Metastasis Reviews, 6:487-501 (1987)].

In addition, the present invention encompasses antibodies that arecapable of binding to the same antigenic determinant as the BR96antibodies and competing with the antibodies for binding at that site.These include antibodies having the same antigenic specificity as theBR96 antibodies but differing in species origin, isotype, bindingaffinity or biological functions (e.g., cytotoxicity). For example,class, isotype and other variants of the antibodies of the inventionhaving the antigen-binding region of the BR96 antibody can beconstructed using recombinant class-switching and fusion techniquesknown in the art [see, e.g., Thammana et al., "Immunoglobulin HeavyChain Class Switch From IgM to IgG In A Hybridoma", Eur. J. Immunol.,13:614 (1983); Spira et al., "The Identification Of Monoclonal ClassSwitch Variants By Subselection And ELISA Assay", J. Immunol. Meth.,74:307-15 (1984); Neuberger et al., "Recombinant Antibodies PossessingNovel Effector Functions", Nature, 312: 604-608 (1984); and Oi et al.,"Chimeric Antibodies", Biotechniques, 4 (3):214-21 (1986)]. Thus, otherchimeric antibodies or other recombinant antibodies (e.g., fusionproteins wherein the antibody is combined with a second protein such asa lymphokine or a tumor inhibitory growth factor) having the samebinding specificity as the BR96 antibodies fall within the scope of thisinvention.

Genetic engineering techniques known in the art are used as describedherein to prepare recombinant immunotoxins produced by fusing antigenbinding regions of antibody BR96 to a therapeutic or cytotoxic agent atthe DNA level and producing the cytotoxic molecule as a chimericprotein.

Examples of therapeutic agents include, but are not limited to,antimetabolites, alkylating agents, anthracyclines, antibiotics, andanti-mitotic agents.

Antimetabolites include methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine.

Alkylating agents include mechlorethamine, thiotepa chlorambucil,melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide,busulfan, dibromomannitol, streptozotocin, mitomycin C, andcis-dichlorodiamine platinum (II) (DDP) cisplatin.

Anthracyclines include daunorubicin (formerly daunomycin) anddoxorubicin (also referred to herein as adriamycin). Additional examplesinclude mitozantrone and bisantrene.

Antibiotics include dactinomycin (formerly actinomycin), bleomycin,mithramycin, and anthramycin (AMC).

Antimytotic agents include vincristine and vinblastine (which arecommonly referred to as vinca alkaloids).

Other cytotoxic agents include procarbazine, hydroxyurea, asparaginase,corticosteroids, mytotane (O,P'-(DDD)), interferons.

Further examples of cytotoxic agents include, but are not limited to,ricin, doxorubicin, taxol, cytochalasin B, gramicidin D, ethidiumbromide, etoposide, tenoposide, colchicin, dihydroxy anthracin dione,1-dehydrotestosterone, and glucocorticoid.

Clearly analogs and homologs of such therapeutic and cytotoxic agentsare encompassed by the present invention. For example, thechemotherapuetic agent aminopterin has a correlative improved analognamely methotrexate.

Further, the improved analog of doxorubicin is an Fe-chelate. Also, theimproved analog for 1-methylnitrosourea is lomustine. Further, theimproved analog of vinblastine is vincristine. Also, the improved analogof mechlorethamine is cyclophosphamide.

4. Immunotoxins of the Invention.

Recombinant immunotoxins, particularly single-chain immunotoxins, havean advantage over drug/antibody conjugates in that they are more readilyproduced than these conjugates, and generate a population of homogenousmolecules, i.e. single peptides composed of the same amino acidresidues.

The techniques for cloning and expressing DNA sequences encoding theamino acid sequences corresponding to the single-chain immunotoxin BR96sFv-PE40, e.g synthesis of oligonucleotides, PCR, transforming cells,constructing vectors, expression systems, and the like arewell-established in the art, and most practitioners are familiar withthe standard resource materials for specific conditions and procedures[see, e.g. Sambrook et al., eds., Molecular Cloning, A LaboratoryManual, 2nd Edition, Cold Spring Harbor Laboratory Press (1989)].

Details of the construction of the single-chain recombinant immunotoxinof the invention, BR96 sFv-PE40 are provided in Example 13, infra.Briefly, polymerase chain reaction (PCR) [Mullis et al., U.S. Pat. Nos.4,683,195 and 4,683,202; Mullis and Faloona, Methods Enzymol.154:335-350 (1987)] is used to amplify a 550 bp BR96 sFv sequence (FIG.35) encoded by plasmid pBR96 Fv using the selected primers.

After PCR amplification and enzymatic digestion the 550 bp fragment isligated using standard procedures into a 4220 bp fragment from vectorpMS8 [Covell et al., Cancer Res. 46:3969-3978 (1986)] encoding the genefor PE40 to form intermediate vector pBW 7.01.

A fragment from pBR96 Fv is then subcloned into pBW 7.01 to form plasmidpBW 7.0 encoding the BR96 sFv-PE40 gene fusion. Correct ligations forvector construction are confirmed by DNA sequence analysis using knownprocedures [Sanger et al., Proc. Natl. Acad. Sci. USA 74:5463 (1977) andMessing et al. Nucleic Acids Res. 9:309 (1981)]. Colonies are thenscreened by restriction enzyme digestion for the appropriate plasmids.

The following include preferred embodiments of the immunoconjugates ofthe invention. Other embodiments which are known in the art areencompassed by the invention. Specific embodiments are set forth in theExamples which follow.

The invention is not limited to these specific immunoconjugates, butalso includes other immunoconjugates incorporating antibodies and/orantibody fragments according to the present invention.

The conjugates comprise at least one drug molecule connected by a linkerof the invention to a targeting ligand molecule that is reactive withthe desired target cell population. The ligand molecule can be animmunoreactive protein such as an antibody, or fragment thereof, anon-immunoreactive protein or peptide ligand such as bombesin or, abinding ligand recognizing a cell associated receptor such as a lectinor steroid molecule.

As previously noted, a conjugate of the invention is represented bygeneral Formula (I): ##STR1##

in which

D is a drug molecule;

n is 1 to 10;

p is 1 to 6;

Y is O or NH₂ ⁺ Cl⁻ ;

z is 0 or 1;

q is about 1 to about 10;

X is a ligand; and,

A is Michael Addition Adduct

For a better understanding of the invention, the drugs and ligands willbe discussed individually. The intermediate used for the preparation ofthe conjugates and the synthesis of the conjugates then will beexplained.

THE DRUG

One skilled in the art understands that the present invention requiresthe drug and ligand to be linked by means of an acylhydrazone linkage,through a Michael Addition Adduct and thioether-containing linker.Neither the specific drug nor the specific ligand is to be construed asa limitation on the present invention. The linkers of the presentinvention may be used with any drug having any desired therapeutic,biological activity-modifying or prophylactic purpose, limited only inthat the drug used in preparing the conjugate be able to form anhydrazone bond. Preferably, to prepare the hydrazone, the drug shouldhave a reactively available carbonyl group, such as, for example, areactive aldehyde or ketone moiety (represented herein as "[D-(C═O)]")which is capable of forming a hydrazone (i.e. a --C═N--NH-- linkage).The drug hydrazone linkage is represented herein as "[D≈N--NH--". Inaddition, the reaction of that reactively available group with thelinker preferably must not destroy the ultimate therapeutic activity ofthe conjugate, whether that activity is the result of the drug beingreleased at the desired site of action or whether the intact conjugate,itself, is responsible for such activity.

One skilled in the art understands that for those drugs which lack areactively available carbonyl group, a derivative containing such acarbonyl group may be prepared using procedures known in the art. As canbe appreciated, the conjugate prepared from such derivatized drug mustretain therapeutic activity when present at the active site, whetherthis is due to the intact conjugate, or otherwise. Alternatively, thederivatized drug or, for example, a prodrug, must be released in such aform that a therapeutically active form of the drug is present at theactive site.

The present linker invention may be used in connection with drugs ofsubstantially all therapeutic classes including, for example,antibacterials, antivirals, antifungals, anticancer drugs,antimycoplasmals, and the like. The drug conjugates so constructed areeffective for the usual purposes for which the corresponding drugs areeffective, and have superior efficacy because of the ability, inherentin the ligand, to transport the drug to the desired cell where it is ofparticular benefit.

Further, because the conjugates of the invention can be used formodifying a given biological response, the drug moiety is not to beconstrued as limited to classical chemical therapeutic agents. Forexample, the drug moiety may be a protein or polypeptide possessing adesired biological activity. Such proteins may include, for example, atoxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin;a protein such as tumor necrosis factor, α-interferon, β-interferon,nerve growth factor, platelet derived growth factor, tissue plasminogenactivator; or, biological response modifiers such as, for example,lymphokines, interleukin-1 ("IL-1"), interleukin-2 ("IL-2"),interleukin-6 ("IL-6"), granulocyte macrophage colony stimulatingfactory ("GM-CSF"), granulocyte colony stimulating factory ("G-CSF"), orother growth factors.

The preferred drugs for use in the present invention are cytotoxicdrugs, particularly those which are used for cancer therapy. Such drugsinclude, in general, alkylating agents, anti-proliferative agents,tubulin binding agents and the like. Preferred classes of cytotoxicagents include, for example, the anthracycline family of drugs, thevinca drugs, the mitomycins, the bleomycins, the cytotoxic nucleosides,the pteridine family of drugs, diynenes, and the podophyllotoxins.Particularly useful members of those classes include, for example,adriamycin, carminomycin, daunorubicin, aminopterin, methotrexate,methopterin, dichloromethotrexate, mitomycin C, porfiromycin,5-fluorouracil, 6-mercaptopurine, cytosine arabinoside, podophyllotoxin,or podophyllotoxin derivatives such as etoposide or etoposide phosphate,melphalan, vinblastine, vincristine, leurosidine, vindesine, leurosineand the like. As noted previously, one skilled in the art may makechemical modifications to the desired compound in order to makereactions of that compound more convenient for purposes of preparingconjugates of the invention.

A highly preferred group of cytotoxic agents for use as drugs in thepresent invention include drugs of the following formulae:

THE METHOTREXATE GROUP OF FORMULA (2): ##STR2##

in which

R¹² is amino or hydroxy;

R⁷ is hydrogen or methyl;

R⁸ is hydrogen, fluoro, chloro, bromo or iodo;

R⁹ is hydroxy or a moiety which completes a salt of the carboxylic acid;

THE MITOMYCIN GROUP OF FORMULA (3): ##STR3##

in which R¹⁰ is hydrogen or methyl;

THE BLEOMYCIN GROUP OF FORMULA (4): ##STR4## in which R¹¹ is hydroxy,amino, C₁ -C₃ alkylamino, di(C₁ -C₃ alkyl)amino, C₄ -C₆ polymethyleneamino, ##STR5## MELPHALAN OF FORMULA (5): ##STR6## 6-MERCAPTOPURINE OFFORMULA (6): ##STR7## A CYTOSINE ARABINOSIDE OF FORMULA (7): ##STR8##THE PODOPHYLLOTOXINS OF FORMULA (8): ##STR9##

in which

R¹³ is hydrogen or methyl;

R¹⁴ is methyl or thienyl;

or a phosphate salt thereof;

THE VINCA ALKALOID GROUP OF DRUGS OF FORMULA (9): ##STR10##

in which R¹⁵ is H, CH₃ or CHO; when R¹⁷ and R¹⁸ are taken singly, R¹⁸ isH, and one of R¹⁶ and R¹⁷ is ethyl and the other is H or OH; when R¹⁷and R¹⁸ are taken together with the carbons to which they are attached,they form an oxirane ring in which case R¹⁶ is ethyl; R¹⁹ is hydrogen,(C₁ -C₃ alkyl)-CO, or chlorosubstituted (C₁ -C₃ alkyl)-CO;

DIFLUORONUCLEOSIDES OF FORMULA (10): ##STR11##

in which R²¹ is a base of one of the formulae: ##STR12##

in which

R²² is hydrogen, methyl, bromo, fluoro, chloro or iodo;

R²³ is --OH or --NH₂ ;

R²⁴ is hydrogen, bromo, chloro or iodo;

THE ANTHRACYCLINES ANTIBIOTICS OF FORMULA (11): ##STR13##

wherein

R₁ is --CH₃, --CH₂ OH, --CH₂ OCO(CH₂)₃ CH₃ or --CH₂ OCOCH(OC₂ H₅)₂

R₃ is --OCH₃, --OH or --H

R₄ is --NH₂, --NHCOCF₃, 4-morpholinyl, 3-cyano-4-morpholinyl,1-piperidinyl, 4-methoxy-1-piperidinyl, benzylamine, dibenzylamine,cyanomethylamine, or 1-cyano-2-methoxyethyl amine

R₅ is --OH, --OTHP, or --H; and,

R₆ is --OH or --H provided that R₆ is not --OH when R₅ is --OH or--OTHP.

The most highly preferred drugs are the anthracycline antibiotic agentsof Formula (11), described previouly. One skilled in the art understandsthat this structural formula includes compounds which are drugs, or arederivatives of drugs, which have acquired in the art different genericor trivial names. Table 14, which follows, represents a number ofanthracycline drugs and their generic or trivial names and which areespecially preferred for use in the present invention.

                                      TABLE 14                                    __________________________________________________________________________                                   1  Formula (1)                                    -                                                                          Compound                                                                             R.sub.1    R.sub.3                                                                           R.sub.4                                                                              R.sub.5                                                                           R.sub.6                                      __________________________________________________________________________    Daunorubicin.sup.a                                                                   CH.sub.3   OCH.sub.3                                                                         NH.sub.2                                                                             OH  H                                              Adriamycin.sup.b CH.sub.2 OH OCH.sub.3 NH.sub.2 OH H                          Detorubicin CH.sub.2 OCOCH(OC.sub.2 H.sub.5).sub.2 OCH.sub.3 NH.sub.2                                        OH H                                           Carminomycin CH.sub.3 OH NH.sub.2 OH H                                        Idarubicin CH.sub.3 H NH.sub.2 OH H                                           Epirubicin CH.sub.2 OH OCH.sub.3 NH.sub.2 H OH                                Esorubicin CH.sub.2 OH OCH.sub.3 NH.sub.2 H H                                 THP CH.sub.2 OH OCH.sub.3 NH.sub.2 OTHP H                                     AD-32 CH.sub.2 OCO(CH.sub.2).sub.3 CH.sub.3 OCH.sub.3 NHCOCF.sub.3 OH       __________________________________________________________________________                                     H                                             .sup.a "Daunomycin" is an alternate name for daunorubicin                     .sup.b "Doxorubicin" is an alternate name for adriamycin                 

Of the compounds shown in Table 14, the most highly preferred drug isadriamycin. Adriamycin (also referred to herein as "ADM") is thatanthracycline of formula (11) in which R₁ is --CH₂ OH, R₃ is --OCH₃, R₄is --NH₂, R₅ --OH, and R₆ is --H.

THE LIGANDS

One skilled in the art understands that "ligand" includes within itsscope any molecule that specifically binds or reactively associates orcomplexes with a receptor or other receptive moiety associated with agiven target cell population. This cell reactive molecule, to which thedrug reagent is linked via the linker in the conjugate, can be anymolecule that binds to, complexes with or reacts with the cellpopulation sought to be therapeutically or otherwise biologicallymodified and, which possesses a free reactive sulfhydryl (--SH) group orcan be modified to contain a such a sulfhydryl group. The cell reactivemolecule acts to deliver the therapeutically active drug moiety to theparticular target cell population with which the ligand reacts. Suchmolecules include, but are not limited to, large molecular weightproteins (generally greater than 10,000 daltons) such as, for example,antibodies, smaller molecular weight proteins (generally, less than10,000 daltons), polypeptide or peptide ligands, and non-peptidylligands.

The non-immunoreactive protein, polypeptide, or peptide ligands whichcan be used to form the conjugates of this invention may include, butare not limited to, transferrin, epidermal growth factors ("EGF"),bombesin, gastrin, gastrin-releasing peptide, platelet-derived growthfactor, IL-2, IL-6, tumor growth factors ("TGF"), such as TGF-α andTGF-β, vaccinia growth factor ("VGF"), insulin and insulin-like growthfactors I and II. Non-peptidyl ligands may include, for example,steroids, carbohydrates and lectins.

The immunoreactive ligands comprise an antigen-recognizingimmunoglobulin (also referred to as "antibody"), or antigen-recognizingfragment thereof. Particularly preferred immunoglobulins are thoseimmunoglobulins which can recognize a tumor-associated antigen. As used,"immunoglobulin" may refer to any recognized class or subclass ofimmunoglobulins such as IgG, IgA, IgM, IgD, or IgE. Preferred are thoseimmunoglobulins which fall within the IgG class of immunoglobulins. Theimmunoglobulin can be derived from any species. Preferably, however, theimmunoglobulin is of human, murine, or rabbit origin. Further, theimmunoglobulin may be polyclonal or monoclonal, preferably monoclonal.

As noted, one skilled in the art will appreciate that the invention alsoencompasses the use of antigen recognizing immunoglobulin fragments.Such immunoglobulin fragments may include. For example, the Fab',F(ab')₂, F_(v) or Fab fragments, or other antigen recognizingimmunoglobulin fragments. Such immunoglobulin fragments can be prepared,for example, by proteolytic enzyme digestion, for example, by pepsin orpapain digestion, reductive alkylation, or recombinant techniques. Thematerials and methods for preparing such immunoglobulin fragments arewell-known to those skilled in the art. See generally, Parham, J.Immunology, 131, 2895 (1983); Lamoyi et al., J. Immunological Methods,56, 235 (1983); Parham, id., 53, 133 (1982); and Matthew et al., id.,50, 239 (1982).

The immunoglobulin can be a "chimeric antibody" as that term isrecognized in the art. Also, the immunoglobulin may be a "bifunctional"or "hybrid" antibody, that is, an antibody which may have one arm havinga specificity for one antigenic site, such as a tumor associated antigenwhile the other arm recognizes a different target, for example, a haptenwhich is, or to which is bound, an agent lethal to the antigen-bearingtumor cell. Alternatively, the bifunctional antibody may be one in whicheach arm has specificity for a different epitope of a tumor associatedantigen of the cell to be therapeutically or biologically modified. Inany case, the hybrid antibodies have a dual specificity, preferably withone or more binding sites specific for the hapten of choice or one ormore binding sites specific for a target antigen, for example, anantigen associated with a tumor, an infectious organism, or otherdisease state.

Biological bifunctional antibodies are described, for example, inEuropean Patent Publication, EPA 0 105 360, to which those skilled inthe art are referred. Such hybrid or bifunctional antibodies may bederived, as noted, either biologically, by cell fusion techniques, orchemically, especially with cross-linking agents or disulfidebridge-forming reagents, and may be comprised of whole antibodies and/orfragments thereof. Methods for obtaining such hybrid antibodies aredisclosed, for example, in PCT application W083/03679, published Oct.27, 1983, and published European Application EPA 0 217 577, publishedApr. 8, 1987, both of which are incorporated herein by reference.Particularly preferred bifunctional antibodies are those biologicallyprepared from a "polydoma" or "quadroma" or which are syntheticallyprepared with cross-linking agents such as bis-(maleimido)-methyl ether("BMME"), or with other cross-linking agents familiar to those skilledin the art.

In addition the immunoglobin may be a single chain antibody ("SCA").These may consist of single chain Fv fragments ("scFv") in which thevariable light ("V_(L) ") and variable heavy ("V_(H) ") domains arelinked by a peptide bridge or by disulfide bonds. Also, theimmunoglobulin may consist of single V_(H) domains (dAbs) which possessantigen-binding activity. See, e.g., G. Winter and C. Milstein, Nature,349, 295 (1991); R. Glockshuber et al., Biochemistry 29, 1362 (1990);and E. S. Ward et al., Nature 341, 544 (1989).

Especially preferred for use in the present invention are chimericmonoclonal antibodies, preferably those chimeric antibodies havingspecificity toward a tumor associated antigen. As used herein, the term"chimeric antibody" refers to a monoclonal antibody comprising avariable region, i.e. binding region, from one source or species and atleast a portion of a constant region derived from a different source orspecies, usually prepared by recombinant DNA techniques. Chimericantibodies comprising a murine variable region and a human constantregion are especially preferred in certain applications of theinvention, particularly human therapy, because such antibodies arereadily prepared and may be less immunogenic than purely murinemonoclonal antibodies. Such murine/human chimeric antibodies are theproduct of expressed immunoglobulin genes comprising DNA segmentsencoding murine immunoglobulin variable regions and DNA segmentsencoding human immunoglobulin constant regions. Other forms of chimericantibodies encompassed by the invention are those in which the class orsubclass has been modified or changed from that of the originalantibody. Such "chimeric" antibodies are also referred to as"class-switched antibodies". Methods for producing chimeric antibodiesinvolve conventional recombinant DNA and gene transfection techniquesnow well known in the art. See, e.g., Morrison, S. L. et al., Proc.Nat'l Acad. Sci., 81, 6851 (1984).

Encompassed by the term "chimeric antibody" is the concept of "humanizedantibody", that is those antibodies in which the framework or"complementarity determining regions" ("CDR") have been modified tocomprise the CDR of an immunoglobulin of different specificity ascompared to that of the parent immunoglobulin. In a preferredembodiment, a murine CDR is grafted into the framework region of a humanantibody to prepare the "humanized antibody". See, e.g., L. Riechmann etal., Nature 332, 323 (1988); M. S. Neuberger et al., Nature 314, 268(1985). Particularly preferred CDR's correspond to those representingsequences recognizing the antigens noted above for the chimeric andbifunctional antibodies. The reader is referred to the teaching of EPA 0239 400 (published Sep. 30, 1987), incorporated herein by reference, forits teaching of CDR modified antibodies.

One skilled in the art will recognize that a bifunctional-chimericantibody can be prepared which would have the benefits of lowerimmunogenicity of the chimeric or humanized antibody, as well as theflexibility, especially for therapeutic treatment, of the bifunctionalantibodies described above. Such bifunctional-chimeric antibodies can besynthesized, for instance, by chemical synthesis using cross-linkingagents and/or recombinant methods of the type described above. In anyevent, the present invention should not be construed as limited in scopeby any particular method of production of an antibody whetherbifunctional, chimeric, bifunctional-chimeric, humanized, or anantigen-recognizing fragment or derivative thereof.

In addition, the invention encompasses within its scope immunoglobulins(as defined above) or immunoglobulin fragments to which are fused activeproteins, for example, an enzyme of the type disclosed in Neuberger, etal., PCT application, WO86/01533, published Mar. 13, 1986. Thedisclosure of such products is incorporated herein by reference.

As noted, "bifunctional", "fused", "chimeric" (including humanized), and"bifunctional-chimeric" (including humanized) antibody constructionsalso include, within their individual contexts constructions comprisingantigen recognizing fragments. As one skilled in the art will recognize,such fragments could be prepared by traditional enzymatic cleavage ofintact bifunctional, chimeric, humanized, or chimeric-bifunctionalantibodies. If, however, intact antibodies are not susceptible to suchcleavage, because of the nature of the construction involved, the notedconstructions can be prepared with immunoglobulin fragments used as thestarting materials; or, if recombinant techniques are used, the DNAsequences, themselves, can be tailored to encode the desired "fragment"which, when expressed, can be combined in vivo or in vitro, by chemicalor biological means, to prepare the final desired intact immunoglobulin"fragment". It is in this context, therefore, that the term "fragment"is used.

Furthermore, as noted above, the immunoglobulin (antibody), or fragmentthereof, used in the present invention may be polyclonal or monoclonalin nature. Monoclonal antibodies are the preferred immunoglobulins,however. The preparation of such polyclonal or monoclonal antibodies nowis well known to those skilled in the art who, of course, are fullycapable of producing useful immunoglobulins which can be used in theinvention. See, e.g., G. Kohler and C. Milstein, Nature 256, 495 (1975).In addition, hybridomas and/or monoclonal antibodies which are producedby such hybridomas and which are useful in the practice of the presentinvention are publicly available from sources such as the American TypeCulture Collection ("ATCC") 12301 Parklawn Drive, Rockville, Md. 20852or, commercially, for example, from Boehringer-Mannheim Biochemicals, P.O. Box 50816, Indianapolis, Ind. 46250.

Particularly preferred monoclonal antibodies for use in the presentinvention are those which recognize tumor associated antigens. Suchmonoclonal antibodies, are not to be so limited, however, and mayinclude, for example, the following:

    ______________________________________                                        Antigen Site                                                                             Monoclonal                                                           Recongnized Antibodies Reference                                            ______________________________________                                        Lung Tumors                                                                              KS1/4       N. M. Varki, et al., Cancer Res.                           44:681, 1984                                                                 534, F8:604A9 F. Cuttitta, et al., in: G. L.                                   Wright (ed) Monoclonal                                                        Antibodies and Cancer Marcel                                                  Dekker, Inc., NY., p. 161, 1984.                                            Squamous Lung Gl, LuCa2, Kyoizumi et al., Cancer Res.,                         LuCa3, LuCa4 45:3274 1985.                                                   Small Cell Lung TFS-2 Okabe et al., Cancer Res.                               Cancer  45:1930, 1985.                                                        Colon Cancer 11.285.14 G. Rowland, et al., Cancer                              14.95.55 Immunol. Immunother., 19:2,                                          NS-3a-22, NS-10 1985 Z. Steplewski, et al.,                                   NS-19-9, NS-33a Cancer Res., 41:2723, 1981.                                   NS-52a, 17-1A                                                                Carcinoembryonic MoAb 35 or Acolla, R.S. et al., Proc. Natl.                   ZCE025 Acad. Sci., (USA), 77:563,                                            Melanoma 9.2.27 1980. T. F. Bumol and R. A.                                     Reisfeld, Proc. Natl. Acad. Sci.,                                             (USA), 79: 1245, 1982                                                       p97 96.5 K. E. Hellstrom, et al.,                                               Monoclonal Antibodies and                                                     Cancer, loc. cit. p. 31.                                                    Antigen T65 T101 Boehringer-Mannheim,                                           P.O. Box 50816,                                                               Indianapolis, IN 46250                                                      Ferritin Antiferrin Boehringer-Mannheim,                                        P.O. Box 50816,                                                               Indianapolis, IN 46250                                                       R24 W. G. Dippold, et al., Proc.                                               Natl. Acad. Sci. (USA),                                                       77:6114, 1980                                                               Neuroblastoma P1 153/3 R. H. Kennet and F. Gilbert,                             Science, 203:1120, 1979.                                                     MIN 1 J. T. Kemshead in Monoclonal                                             Antibodies and Cancer, loc. cit.                                              p. 49.                                                                       UJ13A Goldman et al., Pediatrics,                                              105:252, 1984.                                                              Gliome 5F7, GE2, CG12 N. de Tribolet, et al., in                                Monoclonal Antibodies and                                                     Cancer, loc. cit. p. 81                                                     Ganglioside L6 I. Hellstrom et al.                                              Proc. Natl Acad. Sci. (U.S.A)                                                 83:7059 (1986); U.S. Pat. Nos.                                                4,906,562, issued March 6, 1990                                               and 4,935,495, issued June 19,                                                1990.                                                                        Chimeric L6 U.S. Ser. No. 07/923,244,                                          filed Oct. 27, 1986, equivalent                                               to PCT Patent Publication,                                                    WO 88/03145, published May 5,                                                 1988.                                                                       Lewis Y BR64 U. S. Ser. Nos. 07/289,635,                                        filed December 22, 1988, and                                                  U. S. Ser. No. 07/443,696, filed                                              Nov. 29, 1989, equivalent to                                                  European Patent Publication,                                                  EP A 0375 562, published June                                                 27, 1990.                                                                   fucosylated BR96, Chimeric U.S. Ser. Nos. 07/374,947,                         Lewis Y BR96 filed June 30, 1989, and                                           U. S. Ser. No. 07/544,246, filed                                              June 26, 1990, equivalent to                                                  PCT Patent Publication, WO                                                    91/00295, published January 10,                                               1991.                                                                       Breast Cancer B6.2, B72.3 D. Colcher, et al., in                                Monoclonal Antibodies and                                                     Cancer, loc. cit. p. 121.                                                   Osteogenic Sarcoma 791T/48, M. J. Embleton, ibid, p. 181                       791T/36                                                                      Leukemia CALL 2 C. T. Teng, et al., Lancet, 1:01,                               1982                                                                         anti-idiotype R. A. Miller, et al., N. Eng.                                    J. Med., 306:517, 1982                                                      Ovarian Cancer OC 125 R. C. Bast, et al., J. Clin.                              Invest., 68:1331, 1981.                                                     Prostrate Cancer D83.21, P6.2, J. J. Starling, et al., in                      Turp-27 Monoclonal Antibodies and                                              Cancer, loc. cit., P. 253                                                   Renal Cancer A6H, DBD P. H. Lange, et al., Surgery,                             98:143, 1985.                                                             ______________________________________                                    

In the most preferred embodiment, the ligand containing conjugate isderived from chimeric antibody BR96, "ChiBR96", disclosed in U.S. Ser.No. 07/544,246, filed Jun. 26, 1990, and which is equivalent to PCTPublished Application, WO 91/00295, published Jan. 10, 1991. ChiBR96 isan internalizing murine/human chimeric antibody and is reactive, asnoted, with the fucosylated Lewis Y antigen expressed by human carcinomacells such as those derived from breast, lung, colon, and ovariancarcinomas. The hybridoma expressing chimeric BR96 and identified asChiBR96 was deposited on May 23, 1990, under the terms of the BudapestTreaty, with the American Type Culture Collection ("ATCC"), 12301Parklawn Drive, Rockville, Md. 20852. Samples of this hybridoma areavailable under the accession number ATCC HB 10460. ChiBR96 is derived,in part, from its source parent, BR96. The hybridoma expressing BR96 wasdeposited, on Feb. 21, 1989, at the ATCC, under the terms of theBudapest Treaty, and is available under the accession number HB 10036.The desired hybridoma is cultured and the resulting antibodies areisolated from the cell culture supernatant using standard techniques nowwell known in the art. See, e.g., "Monoclonal Hybridoma Antibodies:Techniques and Applications", Hurell (ed.) (CRC Press, 1982).

In another highly preferred embodiment the immunoconjugate is derivedfrom the BR64 murine monoclonal antibody disclosed in U.S. Ser. No.07/289,635, filed Dec. 22, 1988, and, U.S. Ser. No. 07/443,696, filedNov. 29, 1989, equivalent to European Published Application, EP A 0 375562, published Jun. 27, 1990. As noted above, this antibody also isinternalizing and is reactive with the Lewis Y antigen expressed bycarcinoma cells derived from the human colon, breast, ovary and lung.The hybridoma expressing antibody BR64 and is identified as BR64 wasdeposited on Nov. 3, 1988, under the terms of the Budapest Treaty, withthe ATCC and is available under the accession number HB 9895. Thehybridoma is cultured and the desired antibody is isolated usingstandard techniques well known in the art, such as those referencedabove.

In a third highly preferred embodiment, an immunoconjugate of theinvention is derived from the L6 murine monoclonal antibody disclosed inU.S. Pat. No. 4,906,562, issued Mar. 6, 1990, and U.S. Pat. No.4,935,495, issued Jun. 19, 1990. L6 is a non-internalizing antibodyactive against a ganglioside antigen expressed by human carcinoma cellsderived from human non-small cell lung, breast, colon or ovariancarcinomas. The hybridoma expressing L6 and identified as L6 wasdeposited under the terms of the Budapest Treaty on Dec. 6, 1984 at theATCC and is available under the accession number HB 8677. The hybridomais cultured and the desired antibody is isolated using the standardtechniques referenced above. A chimeric form of the L6 antibody, ifdesired, is described in U.S. Ser. No. 07/923,244, equivalent to PCTPublished Application, WO 88/03145, published May 5, 1988.

Thus, as used "immunoglobulin" or "antibody" encompasses within itsmeaning all of the immunoglobulin/antibody forms or constructions notedabove.

THE INTERMEDIATES AND THE CONJUGATES

The invention provides as intermediates a Michael Addition Receptor- andacylhydrazone-containing drug derivative of Formula (IIa): ##STR15## inwhich D is a drug moiety, n is an integer from 1 to 10 and R is aMichael Addition Receptor, all of which are as defined above.

An especially preferred intermediate encompassed by Formula (IIa) andwhich is useful for preparation of a conjugate of the invention is onedefined by Formula (IIb): ##STR16##

in which

R₁ is --CH₃, --CH₂ OH, --CH₂ OCO(CH₂)₃ CH₃ or --CH₂ OCOCH(OC₂ H₅)₂ ;

R₃ is --OCH₃, --OH or hydrogen;

R₄ is --NH₂, --NHCOCF₃, 4-morpholinyl, 3-cyano-4-morpholinyl,1-piperidinyl, 4-methoxy-1-piperidinyl, benzylamine, dibenzylamine,cyanomethyl amine or 1-cyano-2-methoxyethyl amine,

R₅ is --OH, --OTHP or hydrogen;

R₆ is --OH or hydrogen, provided that R₆ is not --OH when R₅ is --OH or--OTHP;

n is an integer from 1 to 10; and,

R is a Michael Addition receptor moiety.

The most preferred intermediate for use in the present invention isdefined by Formula (IIc): ##STR17## in which R₁, R₃, R₄, R₅ and R₆ areas defined above for Formula (IIb).

Also used as an intermediate in the invention is a targeting ligandwhich contains a freely reactive sulfhydryl group. The sulfhydryl groupcan be contained within the native targeting ligand or can be deriveddirectly from the ligand or from a derivatized form of the ligand. Inthe preferred method for preparing the conjugates of the invention, asulfhydryl group on the ligand or modified ligand reacts directly withthe Michael Addition Receptor of intermediate of Formula (IIa) to formthe final conjugate. Using this process, generally between about one andabout ten drug molecules may be linked to each ligand. Thus, in Formula(I), q may be from about 1 to about 10.

When the conjugate is formed, the Michael Addition Receptor portionbecomes a "Michael Addition Adduct", as used herein. Thus, for example,as one skilled in the art will appreciate, if the Michael AdditionReceptor moiety in the Formulae (IIa) or (IIb) compound is a maleimidomoiety, the corresponding "Michael Addition Adduct" portion of the finalconjugate of Formula (I) will be a succinimido moiety. Thus, a "MichaelAddition Adduct" refers to a moiety which would be obtained had aMichael Addition Receptor, as defined in more detail below, undergone aMichael Addition reaction.

One skilled in the art understands that in the synthesis of compounds ofthe invention, one may need to protect or block various reactivefunctionalities on the starting compounds and intermediates while adesired reaction is carried out on other portions of the molecule. Afterthe desired reactions are complete, or at any desired time, normallysuch protecting groups will be removed by, for example, hydrolytic orhydrogenolytic means. Such protection and deprotection steps areconventional in organic chemistry. One skilled in the art is referred toProtective Groups in Organic Chemistry, McOmie, ed., Plenum Press, N.Y.,N.Y. (1973); and, Protective Groups in Organic Synthesis, Greene, ed.,John Wiley & Sons, New York, N.Y. (1981); for the teaching of protectivegroups which may be useful in the preparation of compounds of thepresent invention.

By way of example only, useful amino-protecting groups may include, forexample, C₁ -C₁₀ alkanoyl groups such as formyl, acetyl, dichloroacetyl,propionyl, hexanoyl, 3,3-diethylhexanoyl, γ-chlorobutyryl, and the like;C₁ -C₁₀ alkoxycarbonyl and C₅ -C₁₅ aryloxycarbonyl groups such astert-butoxycarbonyl, benzyloxycarbonyl, allyloxycarbonyl,4-nitrobenzyloxycarbonyl and cinnaemoyloxycarbonyl; halo-(C₁-C₁₀)-alkoxycarbonyl such as 2,2,2-trichloroethoxycarbonyl; and C₁ -C₁₅arylalkyl and alkenyl groups such as benzyl, phenethyl, allyl, trityl,and the like. Other commonly used amino-protecting groups are those inthe form of enamines prepared with β-keto-esters such as methyl or ethylacetoacetate.

Useful carboxy-protecting groups may include, for example, C₁ -C₁₀ alkylgroups such as methyl, tert-butyl, decyl; halo-C₁ -C₁₀ alkyl such as2,2,2-trichloroethyl, and 2-iodoethyl; C₅ -C₁₅ arylalkyl such as benzyl,4-methoxybenzyl, 4-nitrobenzyl, triphenylmethyl, diphenylmethyl, C₁ -C₁₀alkanoyloxymethyl such as acetoxymethyl, propionoxymethyl and the like;and groups such as phenacyl, 4-halophenacyl, allyl, dimethylallyl,tri-(C₁ -C₃ alkyl)silyl, such as trimethylsilyl,β-p-toluenesulfonylethyl, β-p-nitrophenyl-thioethyl,2,4,5-trimethylbenzyl, β-methylthioethyl, phthalimidomethyl,2,4-dinitrophenylsulphenyl, 2-nitrobenzhydryl and related groups.

Similarly, useful hydroxy protecting groups may include, for example,the formyl group, the chloroacetyl group, the benzyl group, thebenzhydryl group, the trityl group, the 4-nitrobenzyl group, thetrimethylsilyl group, the phenacyl group, the tert-butyl group, themethoxymethyl group, the tetrahydropyranyl group, and the like.

In general, the intermediate Michael Addition Receptor containinghydrazone drug derivative of Formulae (IIa), (IIb), or (IIc) may beprepared, depending on the Michael Addition Receptor moiety used, byreaction of the drug (or derivatized drug) with a hydrazide containing aMichael Addition Receptor in the general manner described in Method A:##STR18##

As noted below, Method A is the preferred method when the MichaelAddition Receptor is a maleimido moiety.

Alternatively, the Formula (IIa) compound may be prepared by reaction ofthe drug with a hydrazide to form an intermediate hydrazone drugderivative followed by reaction of this compound with a Michael AdditionReceptor containing moiety according to the general process described inMethod B: ##STR19##

In Method A and Method B, D, n and R have the meanings previously noted.In Method B, L represents a leaving group, such as for example, halogenmesylate or tosylate, capable of undergoing nucleophilic displacementwhile C represents a group which renders the Michael Addition Receptor,R, a good nucleophilic reagent. Particularly useful groups representedby C may include, for example, alkali metal ions such as Na⁺, K⁺ or Li⁺.

A "Michael Addition Receptor", as one skilled in the art willunderstand, is a moiety capable of reacting with a nucleophilic reagentso as to undergo a nucleophilic addition reaction characteristic of aMichael Addition reaction. As noted, after the nucleophilic additionoccurs, the Michael Addition Receptor moiety is referred to as a"Michael Addition Adduct."

Michael Addition Receptors generally used in the Method A process mayinclude, for example, α,β-ethylenic acids or α,β-thioacids such as thosecontaining a --C═C--COOH, --C═C--C(O)SH, --C═C--C(S)SH, or a--C═C--C(S)OH moiety, α,β-ethylenic esters or thioesters where the alkylmoiety is other than methyl or ethyl, for example, those which contain a--C═C--COOR, --C═C--C(S)OR, --C═C--C(S)SR, or --C═C--C(O)--SR moiety,wherein R is an ester forming group other than methyl or ethyl;α,β-ethylenic amides, imides, thioamides and thiomides (whether cyclicor acylic), for example, those which contain a moiety such as--C═C--CONR₂, --C═--CONHCO--, --C═C--CSNR₂, --C═C--CSNHCO--, or--C═C--CSNHCS--, whether cyclic or acyclic and in which --CONR₂ or--CSNR₂ represents a primary, secondary, or tertiary amide or thioamidemoiety; α,β-acetylenic acids or thioacids, for example, those containinga moiety such as --C.tbd.C--COOH, --C.tbd.C--C(S)OH, --C.tbd.C--C(S)SH,or --C.tbd.C--C(O)--SH; α,β-acetylenic esters, for example those whichcontain a moiety such as --C.tbd.C--COOR, --C.tbd.C--C(S)OR,--C.tbd.C--C(S)SR, or --C.tbd.C--C(O)--SR in which R is an ester forminggroup other than methyl or ethyl; α,β-ethylenic nitriles, for examplethose containing a moiety such as --C═C--C.tbd.N; Michael Additionreactive cyclopropane derivatives, for example, 1-cyano-1-ethoxycarbonylcyclopropane ##STR20## a vinyl dimethyl-sulphonium bromide, for example,one containing a --C═C--S(Me)₂ Br moiety; an α,β-ethylenic sulfone, forexample, one containing a ##STR21## moiety, α,β-ethylenic nitrocompounds, for example, one containing a --C═C--NO₂ moiety;α,β-ethylenic phosphonium compounds, for example one containing a##STR22## group; a compound containing a grouping such as C═C--C═N, aswould be found, for example, in an aromatic heterocycle such as a 2- or4-vinyl pyridine; or a compound containing an α,β-unsaturated thioniumion moiety, such as ##STR23##

Michael Addition Receptors used in Method B may include α,β-ethylenicaldehydes, for example those compounds containing a --C═C--CHO moiety;α,β-ethylenic ketones, for example those compounds containing a##STR24## moiety; α,β-ethylenic esters or thio-esters such as compoundscontaining a --C═C--COOR, --C═C--C(S)OR, --C═C--C(S)SR, or--C═C--C(O)--SR moiety in which R is an ester-forming moiety which ismethyl or ethyl, e.g. ##STR25## α,β-acetylenic aldehydes or ketones, forexample compounds containing a --C.tbd.C--CHO or --C.tbd.C--CO-- moiety;α,β-acetylenic esters or thio- esters that have methyl or ethyl as theiralkyl moiety, for example a compound containing a --C.tbd.C--COOR,--C.tbd.C--C(S)OR, --C.tbd.C--C(O)SR or --C.tbd.C--CSSR group in which Ris an ester forming moiety which is methyl or ethyl.

One skilled in the art may be familiar with other Michael AdditionReceptors which may be used in the present invention. For a generaldiscussion of the Michael Addition Reaction, the reader is referred toE. D. Bergman, D. Ginsberg, and R. Pappo, Org. React. 10, 179-555(1959); and, D. A. Oare and C. H. Heathcock, Topics in Stereochemistry,Vol. 20, eds., E. L. Eliel and S. H. Wilen, John Wiley and Sons, Inc.(1991), and reference cited therein.

The precise reaction conditions used to prepare the intermediates ofFormulae (IIa), (IIb), or (IIc) will depend upon the nature of the drugand the Michael Addition Receptor used in the reaction. The mostpreferred intermediate of the invention is that represented by Formula(IIc), above, in which the drug moiety is an anthracycline drug and theMichael Addition Receptor in a maleimido group. As noted earlier, orthis reaction, Method A, described above, is used. Upon reaction withthe ligand (thiolated, modified or otherwise), the maleimido MichaelAddition Receptor of the intermediate becomes a succinimido group (the"Michael Addition Adduct") in the final conjugate.

The sulfhydryl containing ligands exist naturally (i.e. the ligand hasnot been modified) or may be produced, for example, (a) by thiolation ofthe ligand by reaction with a thiolating reagent such as SMCC orN-succinimid-yl-3-(2-pyridyldithio) propionate ("SPDP") followed byreduction of the product; (b) thiolation of the native ligand byreaction with iminothiolane ("IMT"); (c) addition of a sulfhydrylcontaining amino acid residue, for example, a cysteine residue, to theligand should the ligand, for example, a protein peptide or polypeptide,fail to have a reactive and available sulfhydryl moiety; or, (d)reduction of a disulfide bond in a native molecule using a reducingagent useful for such purposes, for example, dithiothreitol ("DTT").Method (d) is the most preferred method for production of sulfhydrylgroups in antibody molecules used in the conjugates of the invention.

If a thiolating reagent such as SPDP or iminothiolane is used to preparea conjugate of the invention, one skilled in the art will appreciatethat a short "spacer" residue will be inserted between the MichaelAddition Receptor moiety and the ligand in the conjugate of Formula (I).In such a case, z will be 1 in the Formula (I) compound. In thesituation in which a free sulfhydryl group on the ligand is useddirectly, for example by use of a DTT reduced ligand (particularly a"relaxed" antibody prepared using for example, DTT), or in which areactive residue, for example, cysteine is inserted into the ligandportion of the molecule, z in Formula (I) will be 0 and a directthioether bond will exist between the binding ligand and the MichaelAddition portion of the molecule.

To form the conjugate, the thiolated ligand, or ligand having a freelyreactive sulfhydryl group, is reacted with the Michael Addition Receptorcontaining hydrazone of Formula (IIa). In general, the reactionconditions must be chosen with regard to the stability of the ligand,the drug and the desired number of drug moieties to be linked to theligand. For example, one skilled in the art will appreciate that theaverage number of drug molecules linked to the ligand can be varied by(1) modifying the amount of the intermediate drug-hydrazone of Formula(IIa) relative to the number of reactive sulfhydryl groups on the ligandmoiety of the conjugate; or, (2)(a) modifying the number of reactivesulfhydryl groups on the ligand by, for example, only partially reducingthe ligand (in the case of a protein, peptide or polypeptide), (b) byinserting a limited number of, for example, cysteine residues to aprotein, peptide or polypeptide, or (c) by limiting the degree ofthiolation using less than maximal amounts of thiolation agents, forexample, SPDP or iminothiolane. Although the --SH titer can be varied,the preferred level of free sulfhydryl groups, particularly for arelaxed antibody, is the maximum obtainable using the particularreagents in question. The degree of variation in the --SH titer iseasily controlled in the relaxed antibody process. For example, FIG. 62shows the effect on --SH titer for antibodies BR64 and chimeric BR96depending on the mole ratio of DTT to ligand, at 37° C., for a 1.5 hourreaction. One skilled in the art will appreciate that different classesor subclasses of immunoglobulins can have different number of disulfidebridges susceptible to reduction by reagents such as DTT. Thus, afurther consideration in determining the desired level of conjugation ofan antibody or antibody fragment is the number of disulfide groupsavailable for reduction to free --SH groups. In general, however, thepreferred conjugate of Formula (I) will have, on the average from agiven reaction, from about 1 to about 10 drug molecules per ligandmolecule. An especially preferred average drug to ligand molar ratio("MR") is about 4 to about 8.

After the reaction of the conjugate is complete, the conjugate may beisolated and purified using commonly known dialysis, chromatographicand/or filtration methods. A final solution containing the conjugatecustomarily may be lyophilized to provide the conjugate in a dry, stableform which can be safely stored and shipped. The lyophilized producteventually can be reconstituted with sterile water or another suitablediluent for administration. Alternatively, the ultimate product may befrozen, for example under liquid nitrogen, and thawed and brought toambient temperature prior to administration.

In a first preferred embodiment the anthracyclic hydrazone of Formula(IIa) is made by reacting the anthracycline with a maleimido-(C₁-C₁₀)-alkyl hydrazide, or a salt thereof. This reaction is outlined inMethod A, described earlier. The reaction generally is carried out intwo steps. First the maleimido-(C₁ -C₁₀)-alkyl hydrazide, or its salt,is prepared. After purification by, for example, chromatography and/orcrystallization, either the free base of the hydrazide or the salt arereacted with the desired anthracycline or anthracyline salt. Afterconcentration of the reaction solution, the maleimido-containinghydrazone reaction product of Formula (IIa) is collected, and ifdesired, purified by standard purification techniques.

The Formula (IIa) hydrazone then is reacted with a sulfhydryl-containingantibody as described earlier. If the antibody is thiolated using, forexample, N-succinimidyl-3-(2-pyridyldithio)propionate ("SPDP"), thethiolation reaction generally is performed in two steps: (1) Reaction ofa free amino group on the antibody with SPDP; and, (2) DTT reduction ofthe SPDP disulfide to yield a free --SH group. In a preferred procedure,in Step (1) of the thiolation reaction, the SPDP/antibody molar ratioranges between about 7.5:1 to about 60:1, depending upon the number ofsulfhydryl groups desired, with a preferred range of about 7.5:1 toabout 30:1, especially for BR64, and preferably about 20:1 for BR96. Thereaction is carried out between about 0° C. and about 50° C., with amost preferred temperature of about 30° C. The reaction may be carriedout at a pH range of between about 6 and about 8 with the most preferredpH being about 7.4. The reduction in Step (2), using preferably DTT, isperformed using a DTT/SPDP molar ratio of between about 2.5:1 to about10:1. The most preferred DTT/SPDP molar ratio is about 5:1 and thenumber of moles of SPDP is that which is added in Step (1) of thereaction. The reaction generally is carried out at about 0° C. to about40° C., preferably 0° C. and is usually complete after about 20 minutes.After dialysis and concentration of the solution of thiolated ligand (anantibody in the most preferred embodiment), the molar concentration ofsulhydryl groups on the ligand is determined and the thiolated ligand isreacted with the desired molar ratio of the hydrazone derivative ofFormula (IIa) relative to the molar amount of reactive sulhydryl groupson the ligand. Preferably, the ratio is at least about 1:1. Thisreaction generally is performed at a temperature of about 0° C. to about25° C., preferably about 4° C. The resulting conjugate then may bepurified by standard methods. This reaction scheme is outlined in FIGS.49a and 49b.

In a second preferred embodiment, the hydrazone of Formula (IIa) is madeas described above. The hydrazone then is reacted, as outlined in FIG.49c, with an antibody which previously has been thiolated withiminothiolane ("IMT"). Thiolation of the ligand (preferably an antibody)with IMT generally is a one step reaction. The IMT/antibody ratio mayrange from between about 30:1 to about 80:1, preferably about 50:1. Thereaction is performed for about 30 minutes to about 2 hours, preferablyabout 30 minutes, at a pH of about 7 to about 9.5, preferably at a pH ofabout 9, at a temperature of about 20° C. to about 40° C., preferablyabout 30° C. The reaction product then is reacted with the hydrazone ofFormula (IIa) at a temperature of about 0° C. to about 25° C.,preferably at about 4° C. and at a pH of about 7 to about 9.5,preferably about 7.4. The conjugate then is purified using methodsstandard in the art, for example, dialysis, filtration, orchromatography.

In a third especially preferred embodiment the intermediate hydrazone ofFormula (IIa) is made as described above. The hydrazone then is reactedwith a ligand, most preferably, an antibody, in which at least onedisulfide group has been reduced to form at least one sulfhydryl group.An especially preferred ligand is a "relaxed antibody", as describedbelow. The preferred reducing agent for preparing a free sulfhydrylgroup is DTT although one skilled in the art will understand that otherreducing agents may be suitable for this purpose.

A "relaxed" antibody, is one in which one or more, or preferably, threeor more, disulfide bridges have been reduced. Most preferably, a relaxedantibody is one in which at least four disulfide bridges have beenreduced. In a preferred process for preparing a relaxed (i.e. reduced)antibody, the reduction, especially with DTT, and the purification ofthe reaction product, is carried out in the absence of oxygen, under aninert atmosphere, for example, under nitrogen or argon. This process, asdescribed in detail below, allows one to carefully control the degree ofreduction. Thus, this process allows one skilled in the art to reproduceat any time the desired level of reduction of a ligand and, therefore,the number of free --SH groups available for preparing a conjugate ofthe invention.

In an alternative procedure, the reaction is carried out under ambientconditions, however, a sufficiently large amount of the reducing agent,preferably DTT, is used to overcome any reoxidation of the reduceddisulfide bonds which may occur. In either case, purification of theproduct, is carried out as soon as possible after the reaction iscomplete and most preferably under an inert atmosphere such as an argonor nitrogen blanket. The preferred method for preparing the freesulfhydryl containing ligand, however, is the process in whichatmospheric oxygen is excluded from the reaction. An antibody producedby either method is referred to as a "relaxed" antibody. The product,however prepared, should be used for subsequent reaction as quickly aspossible or stored under conditions which avoid exposure to oxygen,preferably under an inert atmosphere.

In the process in which oxygen is excluded from the reaction (i.e. thereaction is performed under an inert atmosphere), the ligand isincubated, for a period of about 30 minutes to about 4 hours, preferablyabout 3 hours, with a molar excess of DTT. The DTT/ligand ratios mayrange between about 1:1 to about 20:1, preferably about 1:1 to about10:1, most preferably about 7:1 to about 10:1, depending upon the numberof sulfhydryl groups desired. For a reduction performed in the presenceof oxygen, the mole ratio of DTT to ligand ranges from about 50:1 toabout 400:1, preferably from about 200:1 to about 300:1. This latterreaction is carried out for about 1 to about 4 hours, preferably 1.5hours, at a temperature of between about 20° C. and about 50° C., with apreferred temperature being about 37° C. The reaction is carried out ata pH of between about 6 and about 8, preferably between about 7 to 7.5.The product then is purified using standard purification techniques suchas dialysis, filtration and/or chromatography. A preferred purificationmethod is diafiltration. To prevent reoxidation of --SH groups, duringpurification and storage, the product preferably is maintained under aninert atmosphere to exclude exposure to oxygen.

One skilled in the art will appreciate that different ligands,particularly an antibody, may possess different degrees ofsusceptibility to reduction and/or reoxidation. Consequently, theconditions for reduction described above may need to be modified inorder to obtain a given reduced ligand such as that described above.Furthermore, alternate means for preparing a reduced antibody useful inthe conjugation process will be evident to one skilled in the art. Thus,however prepared, a reduced ligand used in the preparation of aconjugate of Formula (I) is meant to be encompassed by the presentinvention.

To prepare a conjugate of Formula (I), as noted earlier, the reducedantibody reaction product is reacted with the hydrazone intermediate ofFormula (IIc). The reaction preferably is performed under an inertatmosphere at a temperature of about 0° C. to about 10° C., preferablyat about 4° C. and at a pH of about 6 to about 8, preferably about 7.4.The immunoconjugate is purified using standard techniques such asdialysis, filtration, or chromatography.

In another embodiment of the invention, an anthracycline of Formula (II)is joined to a ligand to which is added a moiety carrying a freesulfhydryl group. In one such embodiment, the ligand is a non-antibodyligand, for example, bombesin. The sulfhydryl may be, for example, partof a cysteine residue added to the native bombesin molecule. Theanthracycline is joined through a hydrazone moiety to a Michael AdditionReceptor containing moiety which then reacts with the modified bombesinto form a conjugate of Formula (I). The product then is purified withstandard techniques such as dialysis, centrifugation, or chromatography.

PREPARATION 1

2,5-Dihydro-2,5-Dioxo-1H-Pyrrolo-1-Hexanoic Acid Hydrazide and itsTrifluoroacetic Acid Salt ("Maleimidocaproyl Hydrazide")

Maleimidocaproic acid (2.11 g, 10 mmol) [See, e.g., D. Rich et al., J.Med. Chem., 18, 1004 (1975); and, O. Keller, et al., Helv. Chim. Acta,58 531 (1975)] was dissolved in dry tetrahydrofuran (200 mL). Thesolution was stirred under nitrogen, cooled to 4° C. and treated withN-methylmorpholine (1.01 g, 10 mmol) followed by dropwise addition of asolution of isobutyl chloroformate (1.36 g, 10 mmol) in THF (10 mL).After 5 min a solution of t-butyl carbazate (1.32 g, 10 mmol) in THF (10mL) was added dropwise. The reaction mixture was kept at 4° C. for ahalf hour and at room temperature for 1 hour. The solvent was evaporatedand the residue partitioned between ethyl acetate and water. The organiclayer was washed with dilute HCl solution, water and dilute bicarbonatesolution, dried over anhydrous sodium sulfate and the solventevaporated. The material was purified by flash chromatography using agradient solvent system of methylene chloride:methanol (100:1-2). Theprotected hydrazide was obtained in 70% yield (2.24 g).

This material (545 mg, 2.4 mmol) was dissolved and stirred intrifluoroacetic acid at 0°-4° C. for 8 min. The acid was removed underhigh vacuum at room temperature. The residue was triturated with etherto yield a crystalline trifluoroacetic acid salt of maleimidocaproylhydrazide (384 mg, 70%). An analytical sample was prepared bycrystallization from methanol-ether, to prepare the product, mp102°-105° C. The NMR and MS were consistent with structure. Anal:Calc'd. for C₁₀ H₁₅ N₃ O₃.0.8CF₃ COOH: C, 44.02; H, 4.99; N, 13.28.Found (duplicate analyses): C, 44.16, 44.13; H, 4.97, 5.00; N, 12.74,12.75.

The salt (220 mg) was converted to the free base by chromatography oversilica using a methylene chloride:methanol:concentrated NH₄ OH(100:5:0.5) solvent system. The material obtained (124 mg, 80%) wascrystallized from methylene chloride-ether to prepare a final product,mp 92°-93° C. NMR and MS were consistent with the structure. Anal:Calc'd, for C₁₀ H₁₅ N₃ O₃ : C, 53.33; H, 6.67; N, 18.67. Found: C,53.12; H, 6.67; N, 18.44.

PREPARATION 2

Maleimidocaproylhydrazone of Adriamycin

A mixture of adriamycin hydrochloride (44 mg, 0.075 mmol),maleimidocaproyl hydrazide (23 mg, 0.102 mmol), prepared according tothe procedure outlined in Preparation 1, and 2-3 drops oftrifluoroacetic acid in absolute methanol (25 mL) was stirred for 15hours under nitrogen and protected from light. At the end of this periodno free adriamycin was detected by HPLC (mobile phase 0.01 molarammonium acetate:acetonitrile, (70:30)). The solution was concentratedat room temperature under vacuum to 10 mL and diluted with acetonitrile.The clear solution was concentration to a small volume, the solid wascollected by centrifugation, and the product was dried under high vacuumto yield the title compound. The NMR was consistent with structure. HighResolution MS, calc'd. for C₃₁ H₄₂ N₄ O₁₃ : 751.2827; Found 751.2804.

The hydrazone also was formed by using adriamycin and thetrifluoroacetic acid salt of the hydrazide. Thus, the salt (40 mg, 0.12mmol), prepared according to the process outlined in Procedure 1, andadriamycin hydrochloride (50 mg, 0.086 mmol) and stirred in methanol (30mL) for 15 hrs. The solution was concentrated to 2 mL and diluted withacetonitrile. The red solid was collected by centrifugation and driedunder vacuum. The product (28 mg, 43%) was identical in NMR and TLC tothe one described above. High Resolution MS calc'd. for C₃₁ H₄₂ N₄ O₁₃ :751.2827; found 751.2819.

5. Expression and Purification of Coding Sequences for BR96 sFv-PE40

The DNA sequences encoding the single-chain immunotoxin may be expressedin a variety of systems as set forth below. The DNA may be excised frompBW 7.0 by suitable restriction enzymes and ligated into suitableprokaryotic or eukaryotic expression vectors for such expression.

To propagate the cloned DNA, the expression plasmid pBW 7.0, encodingthe single-chain immunotoxin, is first transformed into suitable hostcells, such as the bacterial cell line E. coli strain BL21 (lambdaDE3)[provided by Dr. Studier, Brookhaven National Laboratories, New York,described by Chaudhary et al., Proc. Natl. Acad. Sci. USA 84:4538-4542(1987)] using standard procedures appropriate to such cells. Thetreatment employing calcium chloride, as described by Cohen, Proc. Natl.Acad. Sci. USA (1972) 69:2110 (1972) or the CaCl₂ method described inSambrook et al. (eds.), Molecular Cloning: A Laboratory Manual, 2ndEdition, Cold Spring Harbor Press, (1989), may be used for prokaryotesor other cells which contain substantial cell wall barriers.

Depending on the host cell used, transformation or transfection isperformed using standard techniques appropriate to such cells. Forexample, transfection into mammalian cells is accomplished usingDEAE-dextran mediated transfection, CaPO₄ co-precipitation, lipofection,electroporation, or protoplast fusion, and other methods known in theart including: lysozyme fusion or erythrocyte fusion, scraping, directuptake, osmotic or sucrose shock, direct microinjection, indirectmicroinjection such as via erythrocyte-mediated techniques, and/or bysubjecting host cells to electric currents. The above list oftransfection techniques is not considered to be exhaustive, as otherprocedures for introducing genetic information into cells will no doubtbe developed.

Expression in prokaryotic cells is preferred. Prokaryotes mostfrequently are represented by various strains of E. coli; however, othermicrobial strains may also be used. Commonly used prokaryotic controlsequences which are defined herein to include promoters fortranscription initiation, optionally with an operator, along withribosome binding site sequences, include such commonly used promoters asthe beta-lactamase (penicillinase) and lactose (lac) promoter systems[Chang et al., Nature 198: 1056 (1977)], the tryptophan (trp) promotersystem [Goeddel et al., Nucleic Acids Res. 8:4057 (1980)] and the lambdaderived P_(L) promoter and N-gene ribosome binding site [Shimatake etal., Nature 292:128 (1981)].

Expression of the single-chain immunotoxin is detected by Coomassiestained SDS-PAGE and immunoblotting using both anti-idiotypic antibodiesthat bind to BR96, and anti-PE antibodies to bind to the PE40-portion ofthe fusion protein.

6. Recovery of Products

The recombinant immunotoxin may be produced along with a signal sequencein cells capable of processing this sequence for secretion. Whensecreted into the medium, the immunotoxin is recovered using standardprotein purification techniques such as anion-exchange andgel-filtration chromatography. Purification may also be performed usingantibodies reactive with the anti-immunoglobulin portion of theimmunotoxin. However, while the procedures are more laborious, it iswithin the means known in the art to purify the molecule from sonicatesor lysates of cells in which it is produced intracellularly in fused ormature form.

In the preferred embodiment described herein, BR96 sFV-PE40 was purifiedusing anion-exchange and gel-filtration chromatographies with fastprotein liquid chromatography (FPLC) as described by Siegall et al.,Proc. Natl. Acad. Sci. USA 85:9738-9742 (1988).

7. Uses

The BR96 antibody of the invention is useful for diagnosticapplications, both in vitro and in vivo, for the detection of humancarcinomas that possess the antigen for which the antibodies arespecific. In vitro diagnostic methods include immunohistologicaldetection of tumor cells (e.g., on human tissue, cells or excised tumorspecimens) or serologic detection of tumor-associated antigens (e.g., inblood samples or other biological fluids).

Immunohistochemical techniques involve staining a biological specimensuch as a tissue specimen with the BR96 antibody of the invention andthen detecting the presence on the specimen of the antibody complexed toits antigen. The formation of such antibody-antigen complexes with thespecimen indicates the presence of carcinoma cells in the tissue.Detection of the antibody on the specimen can be accomplished usingtechniques known in the art such as immunoenzymatic techniques, e.g.,the immunoperoxidase staining technique or the avidin-biotin (ABC)technique, or immunofluorescence techniques [see, e.g., Ciocca et al.,"Immunohistochemical Techniques Using Monoclonal Antibodies", Meth.Enzymol., 121:562-79 (1986); Hellstrom et al., "Monoclonal MouseAntibodies Raised Against Human Lung Carcinoma", Cancer Research,46:3917-23 (1986); and Kimball (ed.), Introduction To Immunology (2ndEd.), pp. 113-117 (Macmillan Pub. Co. 1986)]. For example,immunoperoxidase staining was used as described in Example 2, infra, todemonstrate the reactivity of the BR96 antibody with lung, breast,colon, and ovary carcinomas and the low reactivity of the antibody withnormal human tissue specimens.

8. Diagnostic Techniques.

Serologic diagnostic techniques involve the detection and quantitationof tumor-associated antigens that have been secreted or "shed" into theserum or other biological fluids of patients thought to be sufferingfrom carcinoma. Such antigens can be detected in the body fluids usingtechniques known in the art such as radioimmunoassays (RIA) orenzyme-linked immunosorbent assays (ELISA) wherein an antibody reactivewith the "shed" antigen is used to detect the presence of the antigen ina fluid sample [see, e.g., Uotila et al., "Two-Site Sandwich ELISA WithMonoclonal Antibodies To Human AFP", J. Immunol. Methods, 42:11 (1981)and Allum et al., supra at pp. 48-51]. These assays, using the BR96antibodies disclosed herein, can therefore be used for the detection inbiological fluids of the antigen with which the BR96 antibodies reactand thus the detection of human carcinoma in patients. Thus, it isapparent from the foregoing that the BR96 antibodies of the inventioncan be used in most assays involving antigen-antibody reactions. Theseassays include, but are not limited to, standard RIA techniques, bothliquid and solid phase, as well as ELISA assays, immunofluorescencetechniques, and other immunocytochemical assays [see, e.g., Sikora etal. (eds.), Monoclonal Antibodies, pp. 32-52 (Blackwell ScientificPublications 1984)].

The invention also encompasses diagnostic kits for carrying out theassays described above. In one embodiment, the diagnostic kit comprisesthe BR96 monoclonal antibody, fragments thereof, fusion proteins orchimeric antibody of the invention, and a conjugate comprising aspecific binding partner for the BR96 antibody and a label capable ofproducing a detectable signal. The reagents can also include ancillaryagents such as buffering agents and protein stabilizing agents (e.g.,polysaccharides). The diagnostic kit can further comprise, wherenecessary, other components of the signal-producing system includingagents for reducing background interference, control reagents or anapparatus or container for conducting the test. In another embodiment,the diagnostic kit comprises a conjugate of the BR96 antibodies of theinvention and a label capable of producing a detectable signal.Ancillary agents as mentioned above can also be present.

The BR96 antibody of the invention is also useful for in vivo diagnosticapplications for the detection of human carcinomas. One such approachinvolves the detection of tumors in vivo by tumor imaging techniques.According to this approach, the BR96 antibody is labeled with anappropriate imaging reagent that produces a detectable signal. Examplesof imaging reagents that can be used include, but are not limited to,radiolabels such as ¹³¹ I, ¹¹¹ In, ¹²³ I, ^(99m) Tc, ³² P, ¹²⁵ I, ³ H,and ¹⁴ C, fluorescent labels such as fluorescein and rhodamine, andchemiluminescers such as luciferin. The antibody can be labeled withsuch reagents using techniques known in the art. For example, see Wenseland Meares, Radioimmunoimaging And Radioimmunotherapy, Elsevier, N.Y.(1983) for techniques relating to the radiolabeling of antibodies [seealso, Colcher et al., "Use Of Monoclonal Antibodies AsRadiopharmaceuticals For The Localization Of Human Carcinoma XenograftsIn Athymic Mice", Meth. Enzymol., 121:802-16 (1986)].

In the case of radiolabeled antibody, the antibody is administered tothe patient, localizes to the tumor bearing the antigen with which theantibody reacts, and is detected or "imaged" in vivo using knowntechniques such as radionuclear scanning using, e.g., a gamma camera oremission tomography [see, e.g., Bradwell et al., "Developments InAntibody Imaging", in Monoclonal Antibodies For Cancer Detection AndTherapy, Baldwin et al. (eds.), pp. 65-85 (Academic Press 1985)]. Theantibody is administered to the patient in a pharmaceutically acceptablecarrier such as water, saline, Ringer's solution, Hank's solution ornonaqueous carriers such as fixed oils. The carrier may also containsubstances that enhance isotonicity and chemical stability of theantibody such as buffers or preservatives. The antibody formulation isadministered, for example, intravenously, at a dosage sufficient toprovide enough gamma emission to allow visualization of the tumor targetsite. Sufficient time should be allowed between administration of theantibody and detection to allow for localization to the tumor target.For a general discussion of tumor imaging, see Allum et al., supra atpp. 51-55.

9. Therapeutic Applications of the Antibodies of the Invention andFragments Thereof.

The properties of the BR96 antibody: a) very high specificity for tumorcells; b) internalization; c) toxicity to antigen-positive tumor cellsalone, i.e. in unmodified form, when used at appropriate concentrations;and d) complement-dependent cytotoxicity and antibody-dependent cellularcytotoxicity activity, suggest a number of in vivo therapeuticapplications. First, the BR96 antibody can be used alone to target andkill tumor cells in vivo.

The antibody can also be used in conjunction with an appropriatetherapeutic agent to treat human carcinoma. For example, the antibodycan be used in combination with standard or conventional treatmentmethods such as chemotherapy, radiation therapy or can be conjugated orlinked to a therapeutic drug, or toxin, as well as to a lymphokine or atumor-inhibitory growth factor, for delivery of the therapeutic agent tothe site of the carcinoma.

Techniques for conjugating such therapeutic agents to antibodies arewell known [see, e.g., Arnon et al., "Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy", in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery", inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review", in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); and Thorpe et al., "The Preparation And CytotoxicProperties Of Antibody-Toxin Conjugates", Immunol. Rev., 62:119-58(1982)].

The BR96 antibody of the invention is particularly suited for use in atherapeutic conjugate because it is readily internalized within thecarcinoma cells to which it binds and thus can deliver the therapeuticagent to intracellular sites of action.

Alternatively, the BR96 antibody can be coupled to high-energyradiation, e.g., a radioisotope such as ¹³¹ I;, which, when localized atthe tumor site, results in a killing of several cell diameters [see,e.g., Order, "Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy", inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985)]. According to yet anotherembodiment, the BR96 antibody can be conjugated to a second antibody toform an antibody heteroconjugate for the treatment of tumor cells asdescribed by Segal in U.S. Pat. No. 4,676,980.

Still other therapeutic applications for the BR96 antibody of theinvention include conjugation or linkage, e.g., by recombinant DNAtechniques, to an enzyme capable of converting a prodrug into acytotoxic drug and the use of that antibody-enzyme conjugate incombination with the prodrug to convert the prodrug to a cytotoxic agentat the tumor site [see, e.g., Senter et al., "Anti-Tumor Effects OfAntibody-alkaline Phosphatase", Proc. Natl. Acad. Sci. USA, 85:4842-46(1988); "Enhancement of the in vitro and in vivo Antitumor Activities ofPhosphorylated Mitomycin C and Etoposide Derivatives by MonoclonalAntibody-Alkaline Phosphatase Conjugates", Cancer Research 49:5789-5792(1989); and Senter, "Activation of Prodrugs by Antibody-EnzymeConjugates: A New Approach to Cancer Therapy," FASEB J. 4:188-193(1990)].

Still another therapeutic use for the BR96 antibody involves use, eitherin the presence of complement or as part of an antibody-drug orantibody-toxin conjugate, to remove tumor cells from the bone marrow ofcancer patients. According to this approach, autologous bone marrow maybe purged ex vivo by treatment with the antibody and the marrow infusedback into the patient [see, e.g., Ramsay et al., "Bone Marrow PurgingUsing Monoclonal Antibodies", J. Clin. Immunol., 8(2):81-88 (1988)].

Furthermore, chimeric BR96, recombinant immunotoxins and otherrecombinant constructs of the invention containing the specificity ofthe antigen-binding region of the BR96 monoclonal antibody, as describedearlier, may be used therapeutically. For example, the single-chainimmunotoxin of the invention, BR96 sFv-PE40 may be used to treat humancarcinoma in vivo.

Similarly, a fusion protein comprising at least the antigen-bindingregion of the BR96 antibody joined to at least a functionally activeportion of a second protein having anti-tumor activity, e.g., alymphokine or oncostatin can be used to treat human carcinoma in vivo.Furthermore, recombinant techniques known in the art can be used toconstruct bispecific antibodies wherein one of the binding specificitiesof the antibody is that of BR96 [see, e.g. U.S. Pat. No. 4,474,893],while the other binding specificity of the antibody is that of amolecule other than BR96.

Finally, anti-idiotypic antibodies of the BR96 antibody may be usedtherapeutically in active tumor immunization and tumor therapy [see,e.g., Hellstrom et al., "Immunological Approaches To Tumor Therapy:Monoclonal Antibodies, Tumor Vaccines, And Anti-Idiotypes", inCovalently Modified Antigens And Antibodies In Diagnosis And Therapy,supra at pp. 35-41].

The present invention provides a method for selectively killing tumorcells expressing the antigen that specifically binds to the BR96monoclonal antibody or functional equivalent. This method comprisesreacting the immunoconjugate (e.g. the immunotoxin) of the inventionwith said tumor cells. These tumor cells may be from a human carcinoma.

Additionally, this invention provides a method of treating carcinomas(for example human carcinomas) in vivo. This method comprisesadministering to a subject a pharmaceutically effective amount of acomposition containing at least one of the immunoconjugates (e.g. theimmunotoxin) of the invention.

In accordance with the practice of this invention, the subject may be ahuman, equine, porcine, bovine, murine, canine, feline, and aviansubjects. Other warm blooded animals are also included in thisinvention.

The present invention also provides a method for curing a subjectsuffering from a cancer. The subject may be a human, dog, cat, mouse,rat, rabbit, horse, goat, sheep, cow, chicken. The cancer may beidentified as a retinoblastoma, papillary cystadenocarcinoma of theovary, Wilm's tumor, or small cell lung carcinoma and is generallycharacterized as a group of cells having tumor associated antigens onthe cell surface. This method comprises administering to the subject acancer killing amount of a tumor targeted antibody joined to a cytotoxicagent. Generally, the joining of the tumor targeted antibody with thecytotoxic agent is made under conditions which permit the antibody sojoined to bind its target on the cell surface. By binding its target,the tumor targeted antibody acts directly or indirectly to cause orcontribute to the killing of the cells so bound thereby curing thesubject.

In accordance with the practice of the invention, the tumor targetedantibody is an internalizing tumor targeted antibody. Examples includeBR96, fragments of BR96, and functional equivalents thereof. Functionalequivalents of BR96 include any molecule which binds the antigen bindingsite to which BR96 is directed and is characterized by (1) bindingcarcinoma cells, (2) internalizing within the carcinoma cells to whichthey bind, and (3) mediating ADCC and CDC effector functions.

Further, in accordance with the practice of the invention, the tumortargeted antibody may be an internalizing tumor targeted antibody whichrecognizes and binds to the Le^(y) determinant. Although, antibodiesdirected against the Le^(y) determinant are known, such antibodies werenot known to internalize within the carcinoma cells to which they bindand/or mediate ADCC and CDC effector functions.

Further, Le^(y) is a fairly common determinant which is overexpressed inmany cancer and some normal cells. Because its presence is widely foundand thus common in both some tumor and non-tumorigenic cells others havequestioned whether such antibodies which recognize Le^(y) may betherapeutically useful.

The claimed invention also provides a method of inhibiting theproliferation of mammalian tumor cells. This method comprises contactingthe mammalian tumor cells with a proliferation inhibiting amount (i.e.effective amount) of a tumor targeted antibody joined to a cytotoxic ortherapeutic agent or anti-tumor drug so as to inhibit proliferation ofthe mammalian tumor cells.

In one example, the tumor targeted antibody is the monoclonal antibodyBR96 produced by hybridoma ATCC HB10036. Other examples includefunctional equivalents of BR96 such as ChiBR96; fragments of BR96;bispecific antibodies with a binding specificity for two differentantigens, one of the antigens being that with which the monoclonalantibody BR96 produced by hybridoma ATCC HB10036 binds; and ahuman/murine recombinant antibody, the antigen-binding region of whichcompetitively inhibits the immunospecific binding of monoclonal antibodyBR96 produced by hybridoma HB 10036 to its target antigen

Also provided is a method of inhibiting the proliferation of mammaliantumor cells which comprises contacting the mammalian tumor cells with asufficient concentration of the immunoconjugate of the invention so asto inhibit proliferation of the mammalian tumor cells.

Examples of such immunoconjugates include, but are not limited to,BR96-PE, PE-BR96 fragment, BR96-RA, BR96 (Fab)-lysPE40, BR96 F(ab')₂-lysPE40, ChiBR96-LysPE40, IL-6-PE40, BR96-DOX.

The subject invention further provides methods for inhibiting the growthof human tumor cells, treating a tumor in a subject, and treating aproliferative type disease in a subject. These methods compriseadministering to the subject an effective amount of the composition ofthe invention.

It is apparent therefore that the present invention encompassespharmaceutical compositions, combinations and methods for treating humancarcinomas. For example, the invention includes pharmaceuticalcompositions for use in the treatment of human carcinomas comprising apharmaceutically effective amount of a BR96 antibody and apharmaceutically acceptable carrier.

The compositions may contain the BR96 antibody or antibody fragments,either unmodified, conjugated to a therapeutic agent (e.g., drug, toxin,enzyme or second antibody) or in a recombinant form (e.g., chimericBR96, fragments of chimeric BR96, bispecific BR96 or single-chainimmunotoxin BR96). The compositions may additionally include otherantibodies or conjugates for treating carcinomas (e.g., an antibodycocktail).

The antibody, antibody conjugates and immunotoxin compositions of theinvention can be administered using conventional modes of administrationincluding, but not limited to, intravenous, intraperitoneal, oral,intralymphatic or administration directly into the tumor. Intravenousadministration is preferred.

The compositions of the invention may be in a variety of dosage formswhich include, but are not limited to, liquid solutions or suspensions,tablets, pills, powders, suppositories, polymeric microcapsules ormicrovesicles, liposomes, and injectable or infusible solutions. Thepreferred form depends upon the mode of administration and thetherapeutic application.

The compositions of the invention also preferably include conventionalpharmaceutically acceptable carriers and adjuvants known in the art suchas human serum albumin, ion exchangers, alumina, lecithin, buffersubstances such as phosphates, glycine, sorbic acid, potassium sorbate,and salts or electrolytes such as protamine sulfate.

The most effective mode of administration and dosage regimen for thecompositions of this invention depends upon the severity and course ofthe disease, the patient's health and response to treatment and thejudgment of the treating physician. Accordingly, the dosages of thecompositions should be titrated to the individual patient. Nevertheless,an effective dose of the compositions of this invention may be in therange of from about 1 to about 2000 mg/m².

The molecules described herein may be in a variety of dosage forms whichinclude, but are not limited to, liquid solutions or suspensions,tablets, pills, powders, suppositories, polymeric microcapsules ormicrovesicles, liposomes, and injectable or infusible solutions. Thepreferred form depends upon the mode of administration and thetherapeutic application.

The most effective mode of administration and dosage regimen for themolecules of the present invention depends upon the location of thetumor being treated, the severity and course of the cancer, thesubject's health and response to treatment and the judgment of thetreating physician. Accordingly, the dosages of the molecules should betitrated to the individual subject.

The interrelationship of dosages for animals of various sizes andspecies and humans based on mg/m² of surface area is described byFreireich, E. J., et al. Cancer Chemother., Rep. 50 (4): 219-244 (1966).Adjustments in the dosage regimen may be made to optimize the tumor cellgrowth inhibiting and killing response, e.g., doses may be divided andadministered on a daily basis or the dose reduced pro- portionallydepending upon the situation (e.g., several divided doses may beadministered daily or proportionally reduced depending on the specifictherapeutic situation.

It would be clear that the dose of the composition of the inventionrequired to achieve cures may be further reduced with scheduleoptimization.

In accordance with the practice of the invention, the pharmaceuticalcarrier may be a lipid carrier. The lipid carrier may be a phospholipid.Further, the lipid carrier may be a fatty acid. Also, the lipid carriermay be a detergent. As used herein, a detergent is any substance thatalters the surface tension of a liquid, generally lowering it.

In one example of the invention, the detergent may be a nonionicdetergent. Examples of nonionic detergents include, but are not limitedto, polysorbate 80 (also known as Tween 80 or (polyoxyethylenesorbitanmonooleate), Brij, and Triton (for example Triton WR-1339 and TritonA-20).

Alternatively, the detergent may be an ionic detergent. An example of anionic detergent includes, but is not limited to, alkyltrimethylammoniumbromide.

Additionally, in accordance with the invention, the lipid carrier may bea liposome. As used in this application, a "liposome" is any membranebound vesicle which contains any molecules of the invention orcombinations thereof.

In order that the invention described herein may be more fullyunderstood, the following examples are set forth. It should beunderstood that these examples are for illustrative purposes only andare not to be construed as limiting the scope of this invention in anymanner.

ADVANTAGES OF THE INVENTION: Initial studies with various previouslyknown immunoconjugates have been disappointing particularly with solidtumors. In our effort to improve antibody based therapy of carcinomas,we have developed and examined novel immunoconjugates and theanti-cancer drug doxorubicin (DOX).

BR96 is important for several reasons. It can trigger irreversiblechanges in membrane structure which leads to tumor cell death, mostlikely through the loss of osmotic control (J. Garrigues, U. Garrigues,I. Hellstrom, K.E. Hellstrom, Am. J. Pathol. 142, 607 (1993)). Further,it is an internalizing MAb that cycles in a nondegraded form between theintracellular compartment and the medium for extended periods of time.The latter characteristic makes BR96 an attractive candidate fortargeting to tumors various agents for selective concentration inantigen positive cells.

The antigen for BR96 is abundantly expressed (>200,000 molecules/cell)on human carcinoma lines. BR96 binds, according to immunohistology, themajority of human carcinomas of the breast, lung and colon. AlthoughBR96, like essentially all MAbs to human tumors, is not trulytumor-specific, it offers advantages over most other antibodies whichrecognize the Le^(y) determinant (K. Lloyd, G. Larson, N. Stromberg, J.Thurin, K. A. Karlsson, Immunogenetics 17, 537 (1983); P. M. Pour, V. E.Tempero, C. Cordon-Cardo, P. Avner, Cancer Res. 48, 5422 (1988); J.Sakamoto et al., ibid. 49, 745 (1989); T. F. Orntoft, H. Wolf, H.Clausen, E. Dabelsteen, S. I. Hakomori, Int. J. Cancer, 43, 774 (1989)).

BR96 is more tumor selective and the normal tissues to which it bindsprimarily comprise differentiated cells of the esophagus, stomach, andintestine as well as acinar cells of the pancreas (Hellstrom I.,Garrigues, H. J. Garrigues, U., Hellstrom, K. E. Cancer Res. 50, 2183(1990)).

BR96 is rapidly internalized into lysosomes and endosomes after bindingto cells expressing the antigen (J. Garrigues et al. 1993).

The antibodies mediate antibody-dependent cellular cytotoxicity"antibody-dependent cellular cytotoxicity", "complement-mediatedcytotoxicity", and "complement-dependent cytotoxicity".

The antibodies can kill antigen-positive tumor cells in the unconjugatedform if present at a sufficient concentration. The antibody conjugatesand recombinant immunotoxins are useful as reagents for killing tumorcells. The antibodies are also useful in diagnostic methods, such as thedetection of carcinomas by in vitro or in vivo technology.

EXAMPLE 1

Preparation Of The BR96 Monoclonal Antibody

The BR96 monoclonal antibody of the invention was produced usinghybridoma fusion techniques as described previously by M. Yeh et al.,Proc. Natl. Acad. Sci. USA, (1979), supra and Yeh et al., Int. J. Cancer(1982), supra. Briefly, a three month-old BALB/c mouse was immunizedusing as the immunogen explanted cultured cells from a human breastadenocarcinoma, designated 3396 or H3396 (from adenocarcinoma of thebreast from a patient which had been established in culture atBristol-Myers Squibb Co., Seattle, Wash.). Methods for establishing andmaintaining cell lines from carcinomas isolated from patients are fullydescribed in Yeh et al., Proc. Natl. Acad. Sci. USA 76:2927-2931 (1979)The mouse received injections on five occasions: on the first fouroccasions, the mouse received one intraperitoneal injection and 1subcutaneous injection split between 4 sites on the mouse. On the fifthoccasion, the mouse was given only one intraperitoneal injection. Thetotal number of cells injected on each occasion was approximately 10⁷cells. Three days after the last immunization, the spleen was removedand spleen cells were suspended in RPMI culture medium. The spleen cellswere then fused with P3-x63-Ag8.653 mouse myeloma cells in the presenceof polyethylene glycol (PEG) and the cell suspension grown in microtiterwells in selective HAT medium as described by Yeh et al., supra [see,also, Kohler and Milstein, Nature, 256:495-97 (1975) and Eur. J.Immunol., 6:511-19 (1976)]. The mixture was seeded to form low densitycultures originating from single fused cells or clones.

The supernatants from these hybridoma cultures were then screened fordirect binding activity on the breast cancer cell line, 3396, and afibroblast cell line obtained from a skin biopsy using an ELISA assaysimilar to that described by Douillard et al., "Enzyme-LinkedImmunosorbent Assay For Screening Monoclonal Antibody Production UsingEnzyme-Labeled Second Antibody", Meth. Enzymol., 92:168-74 (1983).

According to this assay, the antigen (with which the antibody beingscreened for is reactive) is immobilized on microtiter plates and thenincubated with hybridoma supernatants. If a supernatant contains thedesired antibody, the antibody will bind to the immobilized antigen andis detected by addition of an anti-immunoglobulin antibody-enzymeconjugate and a substrate for the enzyme which leads to a measurablechange in optical density. In the present studies, breast cancer cellsor control fibroblast cells were dispensed into a 96-well tissue cultureplate (Costar Cambridge, Mass.) and incubated overnight in a humid 37°C. incubator (5% CO₂). The cells were then fixed with 100 μl of freshlyprepared 1.0% glutaraldehyde to a final well concentration of 0.5% andincubated for 15 min at room temperature, followed by washing threetimes with 1 X phosphate buffered saline (PBS). The cells were nextblocked for 30 min with 5% bovine serum albumin (BSA) in PBS and washedagain three times with PBS. The supernatants from the hybridoma cultureswere then added at 100 μl/well, the wells incubated for 1 h at roomtemperature, and the cells washed three times with PBS. Next, goatanti-mouse horseradish peroxidase (Zymed, Calif.) diluted in 0.1% BSAand PBS was added to a concentration of 100 μl/well. The reactionmixture was incubated for either 1 h at room temperature or 30 min at37° C. and the cells were then washed three times with PBS.o-Phenylenediamine (OPD) was then added at 100 μl/well and the platesincubated in the dark at room temperature for 5-45 min. Antibody bindingto the cells was detected by a color change in the wells that occurredwithin 10-20 min. The reaction was stopped by adding 100 μl/well H₂ SO₄and the absorbance read in a Dynatech (Alexandria, Va.) Microelisaautoreader at 490 nm.

It should be noted that this assay can be performed using intact cellsor purified soluble antigen or cellular extracts as the immobilizedantigen. When soluble antigen or cell extracts were used as antigen, theantigen was initially plated at 50 μl/well in PBS and the plates wereincubated overnight at room temperature before beginning the assay. Whenusing intact cells as antigen, they may be used fresh or after fixation.In either case, the cells were initially plated at 10⁴ cells in 100μl/well in culture medium and incubated overnight in a 37° C. incubator(5% CO₂).

Hybridomas which produced antibodies binding to the breast cancer cellline and not to the human fibroblast cells were thus selected, andtested in a FACS cell sorter on peripheral blood leukocytes (PBLs), asdescribed in Example 2, infra. Hybridomas that were negative on PBLswere cloned, expanded in vitro, and further tested for antibodyspecificity. Those hybridomas producing antibody reactive with humanbreast cancer were recloned, expanded, and injected into pristane-primed3-month old BALB/c mice, where they grew as ascites tumors.

Following this procedure, hybridoma cell line BR96 was obtained, clonedand injected into mice to develop as an ascites tumor. As disclosedabove, the BR96 hybridoma has been deposited with the ATCC. MonoclonalBR96 antibody was purified from ascites by affinity chromatography onimmobilized recombinant protein A (Repligen, Cambridge, Mass.).Clarified ascites was diluted with an equal volume of binding buffer (1M potassium phosphate, pH 8) and applied to a protein A columnpreviously equilibrated with binding buffer. The column was extensivelywashed with binding buffer and then the antibody was eluted with 50 mMphosphoric acid, pH 3. The purified antibody fraction was neutralizedwith 1 M Tris, pH 9 and then dialyzed against phosphate buffered saline.Purified BR96 was finally sterile filtered and stored refrigerated orfrozen.

EXAMPLE 2

Characterization Of The BR96 Monoclonal Antibody

Isotype Determination

To determine the class of immunoglobulin produced by the BR96 hybridoma,the following techniques were utilized:

(a) Ouchterlony Immunodiffusion

An aliquot of supernatant of the hybridoma cells was placed into thecenter well of the a 25 agar plate. Monospecific rabbit anti-mouse Igisotype antibodies (Southern Biotechnology, Birmingham, Ala.) wereplaced in the outer wells and the plate was incubated for 24-28 h atroom temperature. Precipitation lines were then read.

(b) ELISA Isotyping

Dynatech Immulon 96-well plates were coated with goat anti-mouse Igantibodies at 1 μg/ml concentration, 50 μl/well in PBS and left coveredovernight at 4° C. The plates were washed with PBS/Tween 20, 0.05% andblocked with medium at 100 μl/well for 1 h at room temperature. Afterwashing the plates, supernatants from the BR96 hybridoma were added andincubated at room temperature for 1 h. After washing with PBS containing2% bovine serum albumin (BSA), plates were incubated at 37° C. for 30min with monospecific rabbit anti-mouse Ig isotype antibodies coupled toperoxidase (Zymed, South San Francisco, Calif.). After further washing,the plates were incubated with 1 mg/ml OPD and 0.03% H₂ O₂ in 0.1 Mcitrate buffer, pH 4.5. Optical density at 630 nm was determined on aDynatec ELISA plate reader.

Based on these procedures, it was determined that the BR96 monoclonalantibody is of the IgG3 isotype.

Characteristics Of The BR96 Monoclonal Antibody

The BR96 antibody shows a high degree of reactivity with a wide range ofcarcinomas and displays only limited reactivity with normal cells. Thiswas shown by experiments involving immunohistological studies on frozentissue sections as well as binding studies using intact cultured cells.

Immunohistology

The peroxidase-antiperoxidase (PAP) technique of L. A. Sternberger asdescribed in Immunochemistry, pp. 104-69 (John Wiley & Sons, New York,1979) and as modified by J. Garrigues et al., "Detection Of A HumanMelanoma-Associated Antigen, p97, In Histological Sections Of PrimaryHuman Melanomas", Int. J. Cancer, 29:511-15 (1982), was used for theimmunohistological studies. The target tissues for these tests wereobtained at surgery and frozen within 4 h of removal using isopentaneprecooled in liquid nitrogen. Tissues were then stored in liquidnitrogen or at -70° C. until used. Frozen sections were prepared, airdried, treated with acetone and dried again [see Garrigues et al.,supra]. Sections to be used for histologic evaluation were stained withhematoxylin. To decrease non-specific backgrounds sections werepreincubated with normal human serum diluted 1/5 in PBS [see Garrigueset al., supra]. Mouse antibodies, rabbit anti-mouse IgG, and mouse PAPwere diluted in a solution of 10% normal human serum and 3% rabbitserum. Rabbit anti-mouse IgG (Sternberger-Meyer Immunochemicals, Inc.,Jarettsville, Md.), was used at a dilution of 1/50. Mouse PAP complexes(Sternberger-Meyer Immunochemicals, Inc.) containing 2 mg/ml ofspecifically purified PAP was used at a dilution of 1/80.

The staining procedure consisted of treating serial sections with eitherspecific antibody, i.e., BR96, or a control antibody for 2.5 h,incubating the sections for 30 min at room temperature with rabbitanti-mouse IgG diluted 1/50 and then exposing the sections to mouse PAPcomplexes diluted 1/80 for 30 min at room temperature. After eachtreatment with antibody, the slides were washed twice in PBS.

The immunohistochemical reaction was developed by adding freshlyprepared 0.5% 3,3'-diaminobenzidine tetrahydrochloride (Sigma ChemicalCo., St. Louis, Mo.) and 0.01% H₂ O₂ in 0.05 M Tris buffer, pH 7.6, for8 min [see Hellstrom et al., J. Immunol., 127:157-60 (1981)]. Furtherexposure to a 1% 0s0₄ solution in distilled water for 20 min intensifiedthe stain. The sections were rinsed with water, dehydrated in alcohol,cleared in xylene, and mounted on slides. Parallel sections were stainedwith hematoxylin.

The slides were each evaluated under code and coded samples were checkedby an independent investigator.

Typical slides were photographed by using differential interferencecontrast optics (Zeiss-Nomarski). The degree of antibody staining wasevaluated as 0 (no reactivity), + (a few weakly positive cells), ++ (atleast one third of the cells positive), +++ (most cells positive), ++++(approximately all cells strongly positive). Because differencesbetween + and 0 staining were less clear cut than between + and ++staining, a staining graded as ++ or greater was considered "positive".Both neoplastic and stroma cells were observed in tumor samples. Thestaining recorded is that of the tumor cells because the stroma cellswere not stained at all or were stained much more weakly than the tumorcells.

Table 1 below demonstrates the immunohistological staining of varioustumor and normal tissue specimens using the BR96 monoclonal antibody. Asthe table clearly demonstrates, the BR96 antibody reacts with a widerange of human carcinoma specimens, does not react with sarcoma anddisplays only infrequent reactivity with melanoma.

Furthermore, it shows only limited reactivity with any of the largenumber of normal human tissues tested. The only reactivity detected withnormal cells was binding to a small subpopulation of cells in thetonsils and in the testis, and to acinar cells in the pancreas, and tocertain epithelial cells of the stomach and esophagus.

                  TABLE 1                                                         ______________________________________                                        Immunoperoxidase Staining of Human Tumors and Normal                            Tissue Specimens with BR96 Monoclonal Antibody                                                     NUMBER POSITIVE/                                         TISSUE TYPE NUMBER TESTED                                                   ______________________________________                                        Tumors                                                                          Lung carcinoma (non-small cell) 14/17                                         Breast carcinoma 17/19                                                        Colon carcinoma 15/18                                                         Ovary carcinoma  4/4                                                          Endometrial carcinoma  2/2                                                    Melanoma  2/5                                                                 Sarcoma  0/5                                                                  Stomach carcinoma  2/2                                                        Pancreatic carcinoma  2/2                                                     Esophagus carcinoma  2/2                                                      Cervical carcinoma  2/2                                                       Normal Tissue                                                                 Lung  0/7                                                                     Spleen  0/5                                                                   Breast  0/2                                                                   Colon  0/7                                                                    Kidney  0/7                                                                   Liver  0/5                                                                    Brain  0/2                                                                    Heart  0/3                                                                    Skin  0/2                                                                     Thyroid  0/2                                                                  Adrenal  0/1                                                                  Ovary  0/2                                                                    Lymph nodes  0/2                                                              Lymphocyte pellet  0/4                                                        Pancreas  2/2 (only acinar cells                                                were positive)                                                              Uterus  0/7                                                                   Retina  0/1                                                                   Testis  2/2 (only small sub-                                                    population of cells                                                           were positive)                                                              Tonsil  2/2 (only small sub-                                                    population of cells                                                           were positive)                                                              Stomach  2/2 (epithelial cells                                                  positive)                                                                   Esophagus  2/2 (epithelial cells                                                positive)                                                                 ______________________________________                                    

The binding of the BR96 antibody to various cultured cell lines was alsoevaluated. Antibody binding to the cell surface of intact cultured cellswas identified either by a direct binding assay with ¹²⁵ I-labeledantibody as described in Brown et al., "Quantitative Analysis OfMelanoma-Associated Antigen p97 In Normal And Neoplastic Tissues", Proc.Natl. Acad. Sci. USA, 78:539-43 (1981), or by direct immunofluorescenceusing a Coulter Epics C fluorescence activated cell sorter (FACS) II[Hellstrom et al., Cancer Res. 46:3917-3923 (1986)].

For binding analyses using a FACS cell sorter, 2×10⁵ to 1×10⁶ culturedcells were aliquoted in 15% fetal bovine serum (FBS) in IMDM media(Gibco, N.Y.) to a total volume of 500 μl/tube. The cells werecentrifuged for 1.5 min on a Serofuge and the supernatant removed. 100μl of the BR96 monoclonal antibody at 10 μl/ml was added to each tube,the contents of which was then mixed and incubated on ice for 30 min.The reaction mixture was washed three times with 500 μl of 15% FBS/IMDMby centrifugation for 1.5 min on the Serofuge (tubes were blotted afterthe third wash). Then, 50 μl of optimized FITC-conjugated goatanti-mouse IgG antibody (Tago, Burlingame, Calif.) diluted 1:25 in 15%FBS/IMDM was added to each tube and the reaction mixture was mixed andincubated for 30 min. The wash step was then repeated and after blottingof the tubes, each pellet was resuspended in 200-500 μl of PBS. Eachsample was run on a Coulter Epics C FACS and the mean fluorescenceintensity (MFI) was determined. From the MFI, the linear fluorescentequivalent (LFE) was determined. The LFE of each test sample divided bythe LFE of a negative control gave a ratio between the brightness ofcells stained by specific versus control antibody. The binding data isshown in Table 2 below.

                  TABLE 2                                                         ______________________________________                                        FACS Analysis of the Binding of BR96 to                                         Various Types of Suspended Cells                                                                       Ratio                                                Cell line (10 μg/ml)                                                     ______________________________________                                        Breast carcinoma  3396     54                                                   Breast carcinoma MCF-7 38                                                     Breast carcinoma 3630 22                                                      Breast carcinoma 3680 22                                                      Lung carcinoma 2987 15                                                        Lung carcinoma 2707 30                                                        Lung carcinoma 2964  2                                                        Lung carcinoma 3655-3 18                                                      Colon carcinoma RCA 34                                                        Colon carcinoma 3619 22                                                       Colon carcinoma 3347  5                                                       Colon carcinoma HCT116  1                                                     Colon carcinoma CB5 27                                                        Colon carcinoma C 30                                                          Colon carcinoma 2600 16                                                       Ovary carcinioma 2633-3 11                                                    Melanoma 2669  1                                                              Melanoma 3606  1                                                              Melanoma 3620  1                                                              T cell lymphoma CEM  1                                                        line                                                                          T cell lymphoma MOLT-4  1                                                     line                                                                          B cell lymphoma P3HR1  1                                                      line                                                                          Peripheral blood leukocytes   1                                             ______________________________________                                    

As Table 2 demonstrates, the BR96 monoclonal antibody reacted with mostbreast, lung and colon carcinoma cell lines but did not react withmelanoma lines or with T or B lymphoma lines nor with normal peripheralblood leukocytes. Scatchard analysis using radiolabeled antibodyindicated that the approximate association constant (K_(a)) of BR96 wascalculated to be 3.6×10⁶ antigen sites/cell for the 3396 line whichbinds BR96.

These data demonstrate that monoclonal antibody BR96 recognize cellsurface antigens abundantly expressed (up to 10⁶ molecules/cell) on themajority of human carcinomas.

EXAMPLE 3

Internalization Of The BR96 Monoclonal Antibody Within Carcinoma Cells

Studies were conducted to measure internalization of the BR96 monoclonalantibody within antigen-positive carcinoma cells. According to oneprocedure, BR96 was conjugated to the ricin A chain toxin to form animmunotoxin, BR96-RA, whose internalization by carcinoma cells was thendetermined. Uptake of the conjugate by the carcinoma cells was assessedby determining to what extent the tumor cells were killed by ricin Achain.

Conjugation of the antibody to the toxin was carried out as follows:Deglycosylated ricin-A chain (Inland Labs, Austin, Tex.) [see, also,Blakey et al., Cancer Res., 47:947-952 (1987)] was treated withdithiothreitol (5 mM) prior to gel filtration on G-25 Sephadex usingPBS, pH 7.2 as eluant. This was added in a 2:1 molar ratio to theantibody in PBS, the antibody having been previously modified withN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) (Pierce, Rockford,Ill.) according to the procedure of Lambert et al., J. Biol. Chem.260:12035-12041 (1985). Reaction was allowed to proceed for 12-24 h atroom temperature, and the solution was then diluted with 1 volume of H₂O. Removal of unconjugated antibody was achieved using Blue SepharoseCL-6B (Pharmacia, Uppsala, Sweden) [see Knowles et al., Anal. Biochem.,160:440-443 (1987)].

The conjugate and excess ricin-A chain were eluted with high salt(10×PBS) and subjected to further purification on Sephacryl-300(Pharmacia) using PBS as eluant. The resulting conjugate was free ofunbound monoclonal antibody or ricin A-chain and consisted mostly of 1:1adducts.

The internalization of BR96-RA by various carcinoma cell lines was thenmeasured using a thymidine uptake inhibition assay. According to thisassay, the inhibition of ³ H-thymidine incorporation into the DNA of thecarcinoma cells (i.e., the inhibition of cell proliferation) is ameasure of the cytotoxic effect of BR96-RA on the cells and thus ameasure of the internalization of the immunotoxin within the cell.

For the assay, carcinoma cells were plated into a 96-well microtiterplate at 1×10⁴ cells/well in 100 μl of IMDM medium with 15% fetal calfserum (FCS). The plates were incubated for 12-18 h at 37° C. to let thecells adhere. Then the media was removed. Plates were kept on ice. TheBR96-RA immunotoxin (100 μl) was then added in log 10 serial dilutions,starting at 10 μg/ml final concentration down to 0.01 μg/ml. Thereaction mixture was incubated for 4 h on ice. The plates were washedand 200 μl/ml media was added and further incubated at 37° C. for 18 h.At this point, 50 μl of ³ H-thymidine was added at 1 μCi/well and theplates incubated for 6 h at 37° C. In a 5% CO₂ incubator. The assayplates were then frozen at -70° C. for at least 1 h and thawed in a geldryer for 15 min. The cells were harvested onto glass fiber filters(Filter Strips, No. 240-1, Cambridge Technology) in plasticscintillation vials using a PHD cell harvester. 3 ml of scintillationcounting liquid was added to the vials and the vials were counted on aBeckman LS3891 beta scintillation counter at 1 minute per sample.

Graphs of the percent inhibition of thymidine incorporation vs.immunotoxin concentration for each cell line tested were plotted and areshown in FIGS. 1-5. In each assay, a control was run. The results of theassay are expressed as a percentage of the ³ [H] thymidine incorporatedby untreated control cells.

FIG. 1 depicts the percent inhibition of thymidine incorporation bycells from the 3396 breast carcinoma cell line caused by internalizationof BR96-RA. Similar results were obtained with the 2707 lung carcinomacell line (FIG. 2) and C colon carcinoma cell line (see FIG. 4). TheBR96-RA was not internalized by HCT 116 cell line (ATCC No. CCL 247), ahuman colon carcinoma cell line that does not bind BR96 (see FIG. 3).FIG. 5 shows no internalization of BR96-RA on 3347, a colon carcinomacell line to which BR96 does not bind; BR6-RA, on the other hand, whichbinds to the 3347 cells, does internalize. This study, therefore,demonstrated not only internalization of the BR96 antibody but theselectivity of the internalization of the BR96 antibody for antigenpositive carcinoma cells.

EXAMPLE 4

Cytotoxicity of Unmodified BR96 Monoclonal Antibody

Three types of experiments were performed to follow up on the unexpectedobservation that monoclonal antibody BR96 appeared to be cytotoxic byitself (i.e., in unmodified state) when tested in a FACS assay. So as toavoid an effect of complement in serum, all sera used were heatinactivated (56° C. for 30 min); in addition, some of the experimentswith FACS analysis (as described below) were performed on cells whichwere grown in serum-free medium and tested in the absence of serum.

First, living suspended cells from a variety of antigen positivecarcinoma lines (3396, 2987, 3619) were treated with monoclonal antibodyBR96. Cells (5×10⁵) were incubated on ice for 30 min with 100 μl of BR96or control monoclonal antibody at a concentration of 60, 30, 15, 7.5 and3.8 μg/ml in culture medium (IMDM, 15% FBS). After washing the cellstwice with culture medium, the cells were suspended in 500 μl medium andstained by adding the dye propidium iodide which stains dead cells[Krishan, Cell Biol. 66:188 (1975); and Yeh, J. Immunol. Methods, 43:269(1981)]. Out of a 1 mg/ml stock solution (in 70% alcohol) 5 μl dye wasadded to cell samples, incubated on ice for 15 min, washed once andfinally suspended in 500 μl medium. The cells were evaluated on aCoulter Epics C FACS, with dead cells being identified by their redfluorescence. The analysis was done on a two-parameter display with logforward lightscatter in the horizontal and log red fluorescence in thevertical display. Computations of cell size versus cell viability wereobtained by applying the Coulter Epics C Quadstat program. Tumor cellswhich could bind BR96 as well as tumor cells not binding BR96 werestudied in parallel. The results are shown in FIG. 6. FIG. 6demonstrates that incubation of cells from any of three antigen-positivecarcinomas with BR96 rapidly killed them. Untreated or antigen-negativecells were not killed.

Second, tumor cells (3396, 3630, 2987, 3619 and HCT 116) were exposed toBR96 (or the control monoclonal antibody) for 18 h at 37° C. In a96-well microtiter plate at 3×10³ cells/well in 150 μl of IMDM mediumcontaining FBS for 66 h after which 50 μl of ³ [H]-thymidine was addedat 1 μCi/well and the plate was incubated for another 6 h at 37° C.Subsequently, it was frozen at -70° C. for at least 1 h and thawed in agel dryer for 15 min, and the cells harvested onto glass fiber filters.The tritiated thymidine assay was then performed as described in thepreceding example, except that the cells and antibodies were incubatedat 37° C. FIG. 7 illustrates the results. BR96 caused an inhibition of[³ H]thymidine incorporation into antigen-positive cell lines, and thiseffect was dose dependent. The antigen-negative cell line HCT116 was notaffected by any concentration of BR96 examined.

Third, using a modification of a procedure described by Linsley et al.[Linsley, et al., "Identification and characterization of cellularreceptors for growth regulator, Oncostatin M", J. Biol. Chem.264:4282-4289 (1989)] a growth inhibition assay was performed. Cellsfrom four different cell lines (HCT116, 2987, 3396 and 3630) were seeded(3×10³) in a volume of 0.1 ml of IMDM with 15% fetal bovine serum (FBS)in 96-well microtiter plates and allowed to attach for 3 h at 37° C.Various concentrations of whole BR96 monoclonal were then added in avolume of 0.1 ml, after which incubation at 37° C. was continued for 72h. Subsequently, the culture medium was removed and the cells werestained by crystal violet (0.1%, in 20% methanol) for 30 min. and washedthree times with PBS. The bound dye was eluted by the addition of 0.1 mlof a solution of 0.1 M sodium citrate, pH 4.2, in 50% ethanol. Sampleswere assayed in triplicate on an ELISA reader measuring the absorbancein the presence of BR96 with the absorbance in untreated samples. Theresults of this procedure are expressed as percentage inhibition of cellgrowth. FIG. 8 illustrates the results. The results of this assay werein agreement with those presented above for the thymidine incorporationassay (FIG. 7).

EXAMPLE 5

Antibody-Dependent Cellular Cytotoxicity Activity of BR96 Antibody

Determination of antibody-dependent cellular cytotoxicity activity ofBR96 monoclonal antibody was performed as described by Hellstrom et al.,Proc. Natl. Acad. Sci. (USA) 82:1499-1502 (1985). Briefly, a short-term⁵¹ Cr-release test that measures the release of ⁵¹ Cr as described byCerrotini et al., Adv. Immunol. 18:67-132 (1974) was used as evidence oftumor-cell lysis (cytotoxicity). Peripheral blood lymphocytes fromhealthy human subjects were separated on Ficoll-Hypaque [Hellstrom etal., Int. J. Cancer 27:281-285 (1981)] to provide effector cells equalto 5% natural killer cell reactivity against SK-MEL-28 cells (ATCC No.HTB 72); 10⁶ cells were labeled by incubation with 100 μCi (1 Ci=37 Gbq)of ⁵¹ Cr for 2 h at 37° C., after which they were washed three times andresuspended in medium. The labeled cells were seeded (2×10⁴ cells perwell in 20 μl) into Microtiter V-bottom plates (Dynatech Laboratories,Alexandria, Va.). Purified antibody BR96 (10 μg/ml, 1 μg/ml, and 0.1μg/ml) was then added, followed by 2×10⁵ lymphocytes per well in 100 μl.The mixtures were incubated for 2 to 4 h after which the plates werecentrifuged at 400×g. The supernatants were removed and theradioactivity in 100 μl samples was measured with a gamma-counter. Therewere two replicates per group; the variation between replicates was lessthan 10%. Several "criss-cross" experiments were done, in which lung (orcolon) carcinoma and melanoma targets were tested in parallel withmonoclonal antibody BR96 and with the antimelanoma monoclonal antibodyMG-22 [Hellstrom et al., Proc. Natl. Acad. Sci. USA, 82:1499-1502(1985)] which do not bind to most carcinoma cells. Controls included theincubation of target cells alone or with either lymphocytes ormonoclonal antibody separately.

Spontaneous release was defined as the counts per minute (cpm) releasedinto the medium from target cells exposed to neither antibodies norlymphocytes, and total release, as the number of counts released fromtarget cells that were osmotically lysed at the end of the assay.Percent cytotoxicity was calculated as: ##EQU1##

Effector cells were characterized by assessing their sensitivity toincubation with anti-serum to the Leu-11b surface marker and guinea pigcomplement, using procedures described by Hellstrom et al., inMonoclonal Antibodies and Cancer Therapy, UCLA Symposia on Molecular andCellular Biology, New Series, eds. Reisfeld & Sell, Liss, New York, Vol27, pp. 149-164 (1985), incorporated herein by reference. This was doneto measure the expression of the Leu-11b marker, which characterizesnatural killer (NK) cells and is expressed by lymphocytes mediatingantibody-dependent cellular cytotoxicity against human melanoma cells inthe presence of monoclonal antibody BR96. The cytotoxicity by effectorcells alone ("natural killer effect") was subtracted from the dataprovided in FIG. 9.

The results shown in FIG. 9 for an antibody concentration of 10 μg/mlindicate that BR96 mediates antibody-dependent cellular cytotoxicityactivity if present in sufficient concentrations and if the target cellsexpress sufficient concentrations of the epitope. The antibody-dependentcellular cytotoxicity activity can be seen at antibody concentrationslower than those at which the antibody is cytotoxic by itself (usuallyaround 20 μg/ml). When antibody BR96 was used alone as a control itproduced 0% killing at the concentrations tested and using the ⁵¹ Crassay. antibody-dependent cellular cytotoxicity activity was only foundwith BR96 antibody-binding cell lines. Thus, cells from five differentcarcinoma lines, which all bound BR96, were killed viaantibody-dependent cellular cytotoxicity at monoclonal antibodyconcentrations down to 0.1 μg/ml, while cells from a sixth line, 2964,which did not bind BR96, were not killed. The requirement for antibodybinding to obtain antibody-dependent cellular cytotoxicity was furtherdemonstrated by the fact that both of the two carcinomas which couldbind a different antibody, L6 (lines 3619 and 2987), were killed by L6via antibody-dependent cellular cytotoxicity, while the others were not.Under the conditions of the assay, BR96 alone caused the release of only1% of the label, even when tested at a concentration of 10 μg/ml.

EXAMPLE 6 Ability of BR96 to Mediate Complement-Mediated Cytotoxicity(Complement-Dependent Cytotoxicity)

Tests to evaluate the ability of monoclonal antibody BR96 to kill tumorcells in the presence of human serum as a source of complement(complement-mediated cytotoxicity or complement-dependent cytotoxicity)were performed similarly to those for the antibody-dependent cellularcytotoxicity tests described in Example 5, supra, except that 100 μl ofhuman serum from normal human subjects as the source of complementdiluted 1:3 to 1:6 was added per microtest well in place of a suspensionof effector cells.

As shown in FIG. 10, complement-dependent cytotoxicity against cellsbinding BR96 was seen at an antibody concentration of 0.1-5.0 μg/ml,while there was no complement-dependent cytotoxicity against the BR96antigen-negative lines HCT116 and 3347. The 3347 cells could, however,be killed when using the L6 monoclonal antibody, which binds to thesecells. Controls were always included in which BR96 was tested in theabsence of complement. No killing by BR96 alone was detected by the ⁵¹Cr release assay. These data show that BR96 gave a cytotoxic effect inthe presence of human serum at concentrations where it is not cytotoxicby itself. (Control antibody gave no complement-dependent cytotoxicity).

EXAMPLE 7 Determination of Reactivity of BR96 to Glycolipids andGlycoproteins

BR96 antibody was tested for reactivity to a variety of immobilizedglycolipid antigens having known carbohydrate structures and syntheticglycoproteins (so called "neoglycoproteins") using an ELISA assay inwhich purified glycolipids and glycoproteins and antibody were used inexcess (Dr. John Magnani, Biocarb, Gaithersburg, Md.; Lloyd et al.,Immunogenetics 17: 537-541 (1983)). Glycolipids were dried from methanolin microtiter wells at 100 ng/well. Synthetic glycoproteins were coatedon the surface of the wells by incubation of glycoprotein diluted to 200ng in phosphate buffered saline (PBS), at pH 7.4/well. Purified BR96 wasassayed at a concentration of 10 μg/ml in 0.01 M Tris-HCl, pH 7.4,containing 1% BSA containing antibodies from ascites were assayed at adilution of 1:100 in the same buffer. At these high concentrations mostbinding interactions are readily detected. Absorbance values werecalculated as the average of duplicate wells. The results of thisanalysis are summarized in FIGS. 11 and 12 showing that BR96 reactedwith Le^(y) and Lex determinant.

These findings indicate that BR96 can bind to a variant form of theLewis Y (Fuc α1-2Galβ1-4(Fucα1-3)GlcNAc) antigen and that fucose α1-3attached to GlcNAc forms a portion of the Le^(y) -related epitoperecognized by BR96. The high tumor specificity of BR96 and ability tointernalize (not previously described for monoclonal antibodies reactivewith the other Le^(y) determinant) suggests that the antibody recognizesa complex epitope, a portion of which includes at least a part of aLe^(y) determinant.

EXAMPLE 8 Preparation and Characterization of BR96 F(ab')₂ Fragments

Murine BR96 (IgG₃) was purified by Protein A affinity chromatographyfrom murine ascites fluid. Briefly, delipidated ascites was passed overa column containing a matrix of immobilized Protein A (RepliGen Corp.,Cambridge, Mass.) previously equilibrated with 1 M potassium phosphate,pH 8.0. Following the passage of ascites, the column was washed withequilibration buffer until no further protein was spectrophotometricallydetected. The bound BR96 was then eluted from the column using 0.1 Mcitrate buffer, pH 3.0. Immediately after elution, the eluate wasneutralized with 1.0 M Tris buffer, pH 9.0, until the pH wasapproximately 7.0. The monoclonal antibody was then dialyzed into PBSand concentrated prior to storage or use.

F(ab')₂ fragments were then generated by digesting purified BR96monoclonal antibody with pepsin according to Lamoyi, "Preparation ofF(ab')₂ Fragments from Mouse IgG of Various Subclasses", Meth. Enzymol.121: 652-663 (1986). Residual whole antibody and Fc fragments wereadsorbed from the reaction mixture by passage over a protein A affinitycolumn. The resulting F(ab')₂ fragment preparations were dialyzedextensively against PBS and sterile filtered.

The BR96 F(ab')₂ fragments preparations were characterized by gelpermeation HPLC, SDS-PAGE and by ELISA on the human breast tumor line3396 (Bristol-Myers Squibb Co., Seattle, Wash.). Gel permeation HPLC wasused to assess the molecular sizes of the proteins comprising theF(ab')₂ preparation. Reproducible chromatograms from differentpreparations indicated that 75-80% of the protein was F(ab')₂. Noprotein was detected at the positions representing higher molecularweight material, such as whole BR96 or protein aggregates. The remaining20-25% of the protein eluted at positions corresponding to inactivatedpepsin and to other smaller non-protein A-binding digestion products.

Nonreducing and reducing SDS-PAGE was used to examine the denaturedmolecular sizes and structural arrangement of the proteins in theF(ab')₂ preparations. A single major band at the position of F(ab')₂(approximately 100 kdal) was typically observed, with no visiblecontaminating whole monoclonal antibody band (160 kdal). Lower molecularweight bands (i.e. less than 100 kdal) representing inactivated pepsinand small digestion products were minimal. Under reducing conditions theexpected results were obtained with the only major bands occurring as adoublet at approximately 25 kdal representing the light chain and theremaining fragmented portion of the heavy chain. No whole heavy chainband was observed.

Functional (binding) activity of the BR96 F(ab')₂ fragments was comparedto that of whole BR96 in an ELISA with 3396 cells supplying the antigen.Binding of BR96 whole antibody or F(ab')₂ fragments to the cells wasdetected with an HRP-conjugated goat anti-murine K light chain reagentas shown in FIG. 13. On a duplicate plate, binding of whole BR96 wasdistinguished from binding of F(ab')₂ fragments by using HRP-conjugatedprotein A which binds to the whole antibody but not the F(ab')₂fragments (FIG. 14).

These results indicate that BR96 F(ab')₂ (lot R0201-1663-03, lot 2)contained a trace amount of whole BR96 antibody. The level ofcontaminating whole antibody can be estimated to be approximately 8trifold dilutions away from the amount of F(ab')₂ present, or about0.01%. The other F(ab')₂ preparation (lot R9011-1481-48, lot 1) showedno detectable level of contaminating whole BR96, indicating that anyeffect of BR96 can be explained by binding of the Fab region and not theFc region.

In summary, the BR96 F(ab')₂ preparations appear to be completely freeof contaminating whole BR96 IgG by HPLC and by SDS-PAGE. In only oneinstance, when a very sensitive ELISA method was used were detectablelevels of contaminating whole BR96 antibody found and this representedonly approximately 0.01% by weight compared to the amount of F(ab')₂fragments present.

EXAMPLE 9 Preparation and Characterization of Chimeric BR96 Antibody(ChiBR96)

The murine/human chimeric BR96 antibody of the invention ("ChiBR96") wasproduced using a two-step homologous recombination protocol as describedby Fell et al., in Proc. Natl. Acad. Sci. USA 86: 8507-8511 (1989) andin co-pending patent application by Fell and Folger-Bruce, U.S. Ser. No.243,873, filed Sep. 14, 1988, and Ser. No. 468,035, filed Jan. 22, 1990,and assigned to the same assignee as the present application; thedisclosures of all of these documents are incorporated in their entiretyby reference herein.

Human Heavy Chain DNA Transfection

The murine hybridoma cell line BR96, ATCC No. HB10036, obtained asdescribed above was transfected (8×10⁶ cells) with hγ1/HC-D (depositedat Agricultural Research Service Culture Collection, Peoria, Ill., NRRLNo. B 18599) (FIG. 15) by electroporation (Gene Pulser; BioradLaboratories, Richmond, Calif.) at 250 V, 960 μFd capacitance setting,in isotonic phosphate buffered saline (PBS) and 30 μg/ml of the purified6.2 kb Xbal restricted fragment of the vector hgγ1HC-D. After 48 hrcells were seeded in 96-well plates at 10⁴ cells/well. Selection forNeo^(R) was carried out in IMDM medium (GIBCO, Grand Island, N.Y.)containing 10% (vol/vol) fetal bovine serum (FBS) and the antibioticaminoglycoside G418 (GIBCO) at 2.0 mg/ml.

Detection of Secreted Human IgG (Hu γ1) Antibody by ELISA

Culture supernatants were screened using a sandwich ELISA assay 2 weeksafter transfection. Goat anti-human IgG, Fc specific (CALTAG, SanFrancisco, Calif.) was used as the capture antibody and goat anti-humanIgG, Fc specific conjugated to horseradish peroxidase HRPO, (CALTAG) wasthe antibody used to detect bound human IgG. Cells from the HuIgGpositive wells were subcloned by dilution and dilution clones werescreened by ELISA to detect human IgGγ1 by the previously describedmethod. The clones containing human IgGγ1 were also screened by ELISA todetect murine IgG3 heavy chain. Goat anti-mouse IgG3 (SouthernBiotechnology Assoc., Inc., Birmingham, Ala.) was used as the captureantibody and goat anti-mouse conjugated to HRPO (Southern BiotechnologyAssoc., Inc.) was the antibody used to detect the mouse IgG3.

One of the human IgGγ1 positive murine IgG3 negative (Huγ1⁺, MuG3⁻)clones was chosen and designated ChiHBR96. This heavy chain chimerichybridoma cell line, ChiHBR96 was characterized for antigen specificityon MCF-7 cells and for expression levels by a quantitative ELISA forhuman IgG expression on MCF7 cells. The cell line ChiHBR96 expressedapproximately 20 μg/ml of antigen-specific human IgG antibody.

Light Chain DNA Transfection

The ChiHBR96 hybridoma (8×10⁶ cells) was transfected by electroporationas described above but using 30 μg/ml of the human light chainrecombination vector pSV₂ gpt/C_(K) (NRRL No. B 18507) containing thehuman light chain K immunoglobulin sequence shown in FIG. 16, linearizedwith HindIII. After 48 hr cells were seeded in 96-well plates at 10⁴cells/well. Selection for gpt was carried out in IMDM medium containing10% (vol/vol) FBS, 15 μg/ml hypoxanthine, 250 μg/ml xanthine and 2.25μg/ml mycophenolic acid (MA).

Detection of Secreted Human Kappa (Hu K) Antibody by ELISA

Culture supernatants were screened using a sandwich ELISA assay asdescribed above, 2 weeks after transfection. Goat α-human K (CALTAG) wasthe capture antibody and goat anti-human K HRPO (CALTAG) was theantibody used to detect bound human K. Wells containing human K antibodywere subcloned by dilution and the clones were screened by ELISA todetect human K or murine K chain. Goat anti-mouse K (Fisher Scientific,Pittsburgh, Pa.) was used as the capture antibody and goat anti-mouse Kconjugated to HRPO (Fisher Scientific) was the antibody used to detectthe presence of the mouse K chain. One of the human K positive, murine Knegative clones (HuK⁺, MuK⁻) was chosen to analyze antigen specificityon MCF-7 cells and for expression levels by a quantitative ELISA forhuman IgG expression on MCF-7 cells. A cell line that was antigenspecific for MCF-7 cells and HuIgG⁺, MuIgG3⁻, HuK⁺, MuK⁻ was chosen anddesignated Chimeric BR96 (ChiBR96).

The original expression of the heavy and light chain antigen specificchimeric BR96 (ChiBR96) antibody was approximately 25 μg/ml. Throughfour sequential rounds of cloning the line in soft agarose with a rabbitα HuIgG antibody overlay to detect cells secreting the highest amount ofchimeric antibody [Coffino et al., J. Cell. Physiol. 79: 429-440(1972)], a hybridoma cell line (ChiBR96) was obtained secretingapproximately 130 μg/ml of chimeric antibody. Hybridoma ChiBR96 wasdeposited with the ATCC on May 23, 1990, and there provided with thedeposit number, ATCC No. HB 10460.

Binding of ChiBR96

The relative affinity of the ChiBR96 antibody and murine BR96 antibodyof the invention for the tumor associated antigen on MCF-7 cells wasdetermined by an ELISA competition binding assay [Hellstrom et al.,Cancer Res. 50: 2449-2454 (1990)]. Briefly, adherent antigen bearingcell line MCF-7 was plated in a 96-well microtiter dish at 3×10⁴cells/well and allowed to grow to confluency for about 3-4 days. Thegrowth media was discarded and the cells are fixed with 0.5%glutaraldehyde in PBS (Sigma Chemical Co., St. Louis, Mo.), at 100μl/well for 30 min. The glutaraldehyde was discarded and the plate waswashed gently with PBS three times. The plate was then blocked withbinding buffer (0.1% BSA in DMEM) 200 μl/well for 1 hr or was storedindefinitely at -20° C. Binding buffer was discarded and samples andstandards were added to the wells. The plates were covered and incubatedovernight at 4° C. Samples and standards were discarded and the plateswere washed three times with PBS. HRP-conjugate diluted in 1% horseserum in PBS was added to wells, 100 μl/well and incubated for 1 hr at37° C. The ELISA was developed with 3,3',5,5'-tetramethyl benzidine(TMB) chromagen (Genetic Systems, Seattle, Wash.) in a citrate buffer.Color development was arrested with 3N H₂ SO₄ and the plate was read ona Titertek Microplate reader at 450 nm. This assay determined how well0.3 μg/ml of biotinylated ChiBR96 antibody competes with eitherunlabeled ChiBR96 or unlabeled murine BR96 monoclonal antibody for theantigen. The bound biotinylated ChiBR96 antibody was detected withavidin-HRPO and developed with standard ELISA reagents.

As shown in FIG. 17, the overlap of the two binding curves indicatesthat the two antibodies have the same specificity and relative affinityfor the tumor antigen.

EXAMPLE 10 Characterization of the ChiBR96 Antibody and BR96 F(ab')₂Fragments

Cytotoxicity of Unmodified ChiBR96 and BR96 F(ab')₂ Fragments

Living suspended cells from the BR96 antigen positive carcinoma lines3396, 2987 and MCF-7, were treated with ChiBR96 and BR96 F(ab')₂fragments prepared as described in Examples 8 and 9, above, to determinecytotoxicity of these antibodies as compared to the BR96 monoclonalantibody of the invention. The cytotoxicity tests were performed by FACSassay as described above in Example 4. The results of these experimentsare shown in FIGS. 18-20 as percentage dead cells vs. antibodyconcentration in μg/ml.

FIGS. 18 and 20 show that the chimeric BR96 antibody and F(ab')₂fragments of BR96 IgG3 are similar to BR96 monoclonal antibody withrespect to cytotoxicity to 3396 and MCF-7 cells. FIG. 19 demonstratesthat the cytotoxic effect on 2987 cells is much lower than on the otherbreast carcinoma cells (FIGS. 18 and 20). These results suggest that ahigher binding ratio (Table 2) is important for killing by theseantibodies and/or that different tumor cells might have differentsensitivity to killing by these antibodies. These results illustratethat the ChiBR96 antibody and the F(ab')₂ fragments are cytotoxic bythemselves, i.e. in unconjugated form, and also illustrate that thecytotoxicity of the BR96 antibodies is not dependent on the Fc region.

Internalization of ChiBR96

The internalization of the ChiBR96 antibody within carcinoma cells wasevaluated in comparison to internalization of the BR96 monoclonalantibody. The antibodies were conjugated to ricin A chain toxin to formimmunotoxins ChiBR96-RA (1-4 Ricin A chains per antibody molecule) andBR96-RA (1-2 Ricin A chains per antibody molecule) and internalizationby carcinoma cell lines 3396 and 3630 was measured using a thymidineuptake inhibition assay, as described in Example 3, above.

Graphs of the percent inhibition of thymidine incorporation vs.immunotoxin concentration for each cell line tested are shown in FIGS.21 and 22. FIG. 21 depicts the percent inhibition of thymidineincorporation by cells from the 3396 breast carcinoma cell line causedby internalization of ChiBR96-RA and BR96-RA. As shown in the graph,ChiBR96 is internalized similarly to BR96, and appears to be at least asefficient as BR96 at killing tumor cells. Similar results were obtainedwith the 3630 breast carcinoma cell line (FIG. 22).

Antibody-dependent cellular cytotoxicity Activity of ChiBR96 Antibody

Determination of antibody-dependent cellular cytotoxicity activity ofChiBR96 was conducted as described in Example 5, above using thefollowing cell lines: breast cancer lines 3396, 3630 and 3680(Bristol-Myers Squibb Co., Seattle, Wash.) and MCF-7 (ATCC No. HTB22);ovarian cancer line 3633-3 (Bristol-Myers Squibb Co., Seattle, Wash.);and lung cancer lines 2987; 3655-3 and 2981 (Bristol-Myers Squibb Co.,Seattle, Wash.). The results are shown in Table 3 for various antibodyconcentrations.

                  TABLE 3                                                         ______________________________________                                        antibody-dependent cellular cytotoxicity Activity of Chi-BR96                                  Antibody                                                       Concentration (μg/ml)                                                    Cell Lines                                                                             Antibody NK     10  1     0.1 0.01   0.001                           ______________________________________                                        Breast Cancer                                                                          BR96     28     86  74    59  27     25                                3396 ChiBR96  88 79 60 34 26                                                  MCF-7 BR96 16 16 82 69 54 15                                                   ChiBR96  90 82 57 25 17                                                      MCF-7 BR96 22 73 69 48 22 22                                                   ChiBR96  76 70 57 33 26                                                      3630 BR96 30 69 64 42 30 34                                                    ChiBR96  69 56 42 36 36                                                      3680 BR96 13 73 67 58 34 38                                                    ChiBR96  70 71 61 39 30                                                      Ovarian Cancer BR96 20 92 90 64 28 23                                         3633-3 ChiBR96  88 88 54 43 29                                                Lung Cancer BR96 11 51 57 41  9  7                                            2987 ChiBR96  69 65 51 28 15                                                  3655-3 BR96  4 49 37  0  0  0                                                  ChiBR96  39 35 12  6  5                                                      2981 BR96  3  4  3  3  4  5                                                    ChiBR96   5  4  3  4  4                                                    ______________________________________                                    

The results shown in Table 3 for various antibody concentrationsindicate that ChiBR96 mediates antibody-dependent cellular cytotoxicityactivity to a similar extent as BR96. The antibody-dependent cellularcytotoxicity activity can be seen at antibody concentrations lower thanthose at which the ChiBR96 antibody is cytotoxic by itself. Whenantibody BR96 was used alone as a control it produced 0% killing at theconcentrations tested. antibody-dependent cellular cytotoxicity activitywas only found with the BR96 antibody-binding cell lines.

Ability of ChiBR96 to Mediate Complement-Mediated Cytotoxicity

Determination of the ability of ChiBR96 to kill tumor cells in thepresence of human serum as a source of complement (complement-dependentcytotoxicity) were performed as described in Example 6, using breastcell lines 3396; MCF-7, 3630 and 3680; ovarian cancer cell line 3633-3;and lung cancer cell lines 3655-3, 2987 and 2981. Table 4 presents theresults.

                  TABLE 4                                                         ______________________________________                                        complement-dependent cytotoxicity Activity of ChiBR96                                           Antibody                                                      Concentration (μg/ml)                                                    Cell Lines  Antibody  10     1      0.1 0.01                                  ______________________________________                                        Breast Cancer                                                                             BR96      100    99     78  13                                      3396 ChiBR96 86 92 13 2                                                       MCF-7 BR96 94 100  63 2                                                        ChiBR96 92 83  1 0                                                           3630 BR96 94 100  82 9                                                         ChiBR96 86 86 33 9                                                           3680 BR96 100  100  19 7                                                       ChiBR96 87 100   5 9                                                         Ovarian Cancer BR96 98 98 21 0                                                3633-3 ChiBR96 100  100  26 1                                                 Lung Cancer BR96 91 22  0 0                                                   3655-3 ChiBR96 46  3  0 0                                                     2987 BR96 100  100   1 0                                                       ChiBR96 100  43  0 0                                                         2981 BR96  0  3  3 2                                                           ChiBR96  1  1  2 10                                                        ______________________________________                                    

As shown in Table 4, ChiBR96 gave a cytotoxic effect(complement-dependent cytotoxicity) similar to that of BR96, in thepresence of human serum containing complement. BR96 and ChiBR96 were notcytotoxic in any concentration. Human serum was also not cytotoxic.

The above results demonstrate that the whole BR96 antibody and chimericantibody of the invention are internalized within carcinoma cells towhich they bind, are cytotoxic alone in unmodified form and haveantibody-dependent cellular cytotoxicity and complement-dependentcytotoxicity activity for cells expressing a higher amount of epitopes.

EXAMPLE 11 Evaluation of BR96 Antibodies In Vivo

The therapeutic potential of the unmodified BR96 antibody of theinvention for treatment of tumors was examined in a series ofexperiments using human tumor xenografts in nude mice. In addition,certain parameters were examined that might influence the efficacy ofBR96 as an antitumor agent. These parameters include level of antigenexpression on the target tumor line, time from tumor implantation toinitiation of therapy and effects of dose.

In all the in vivo experiments of this example, the required number ofBalb/c nu/nu mice (Harlan Sprague Dawley, Indianapolis, Ind.) wereimplanted with either the human lung adenocarcinoma cell line H2987 orH2707 tumor line. Cells from these tumor lines were grown in vitro,harvested, washed and resuspended in PBS prior to subcutaneous (s.c.)implantation of 10 million cells into the rear flank of each mouse.These groups of mice were then randomized and separated into smallerequal groups of 8 or 10 mice each.

To increase the chance of observing any antitumor effects of BR96 whilestill requiring the antibody to actually localize to the tumor implantsite for any effect to occur, therapy was initiated 24 hours after tumorimplantation on day 2. Both the BR96 and control monoclonal antibodieswere administered at the same dose and schedule, although initiation oftherapy in some cases varied. The treatment dose was administered in 0.2ml PBS intravenously (i.v.) through the tail vein of the mouse. Normallythe schedule was once every three days for five injections (Q3DX5).However, two extra injections were given on days 19 and 21 after H2987tumor implantation in the initial experiment.

Antitumor Effects of BR96 Antibody in 2987 and 2707 Tumors

Tumor volumes were determined for each animal weekly with measurementusually beginning on the eighth day after implantation. Tumor volumeswere calculated from the measurements of tumor length and perpendicularwidth using the formula:

    Tumor Volume=longest length×(perpendicular width squared/2)

Group mean values were then calculated and plotted against time aftertumor implantation.

In the initial experiment depicted in FIG. 23 treatment with BR96resulted in highly significant anti-tumor effects against the H2987 cellline. BR64, which also binds and is internalized by these cells, wasused as a negative control, and showed little if any effect compared tothe PBS treated controls.

Table 5 summarizes the effects on the individual tumors at the end oftreatment in this first experiment.

                  TABLE 5                                                         ______________________________________                                        Effects of Treatment with Unmodified BR96 Initiated                             At Different Times After H2987 Implantation                                   EXPERIMENT 1                                                                  DAY 28                                                                                TUMOR RESPONSE          PRO-                                        GROUP MAb     COMPLETE  PARTIAL                                                                              STABLE GRESSION                                ______________________________________                                        1     BR96    2         0      3      5                                         2 BR64 0 0 1 9                                                                3 PBS 0 0 0 10                                                              ______________________________________                                    

Only treatment with BR96 antibody resulted in complete absence of tumor.Two animals in this group were tumor free and an additional 3 animalsshowed cessation of growth of their tumors following treatment with BR96antibody. The two mice showing no signs of tumor remained tumor freethroughout the course of the experiment.

Antitumor Effects of BR96 Antibody on Established Tumors

One of the ultimate goals of tumor therapy is the effective treatment ofestablished and growing tumors. To examine whether BR96 could have anantitumor effect on established tumors the H2987 or H2707 lungadenocarcinoma tumor lines were used as xenografts in nude mice. Becauseboth of these tumor lines result in palpable tumors eight days afteradministration of 10 million cells s.c., delaying initiation oftreatment provided a method to examine antitumor effects on establishedtumors.

Therefore, to further examine the efficacy of unmodified BR96, severalexperiments were performed where treatment was withheld for either 5 or8 days following s.c. tumor implantation. The delay in treatmentinitiation allowed the tumor cells to become established tumors. Thisresults in an animal model that is more difficult to treat but resemblesthe clinical situation in a more realistic manner.

The treatment protocol is summarized in Table 6. Three groups of 10 miceeach were treated with BR96 antibody initiated at different times asdescribed in this Table. Control mice received either FA6 or PBSbeginning on DAY 2. FA6 is a murine IgG₃ directed against a bacterialantigen not found in mammalian species, and acted as an isotype matchednonbinding negative control monoclonal antibody.

                  TABLE 6                                                         ______________________________________                                        Effects of Treatment With Unmodified BR96                                       Initiated at Different Times After H2987 or                                   H2707 Implantation                                                            TREATMENT PROTOCOL                                                            GROUP   MAb     SCHEDULE/ROUTE                                                                            DOSE  DAYS INJECTED                             ______________________________________                                        1     BR96    Q3D × 5 i.v.                                                                          1 mg  2, 5, 8, 11, 14                               2 BR96 Q3D × 5 i.v. 1 mg 5, 8, 11, 14, 17                               3 BR96 Q3D × 5 i.v. 1 mg 8, 11, 14, 17, 20                              4 FA6 Q3D × 5 i.v. 1 mg 2, 5, 8, 11, 14                                 5 PBS Q3D × 5 i.v. 0.2 ml 2, 5, 8, 11, 14                             ______________________________________                                    

The results of this treatment protocol for both H2987 and H2707 tumorcell lines are shown in FIG. 24, where the number of animals withouttumors versus when initiation of treatment after tumor implantationoccurred are plotted. Absence of tumor, as defined by the absence of apalpable tumor, was assessed at the end of treatment for each group. Theday used for the determination of tumor absence varied since treatmentwas initiated at different times post tumor implant. Early initiation oftreatment was clearly more effective and efficacy decrease as onset oftreatment increased from time of tumor implant. Since delay ininitiation of treatment allows greater growth and establishment of thetumor, decreased efficacy at later treatment initiation times reflectsthe increasing difficulty of treating larger and more establishedtumors.

These results demonstrate that BR96 has antitumor effects against twodifferent tumor cell lines. Antitumor effects were only observed in thethree groups treated with BR96 antibody while those animals treated witheither the control FA6 or PBS showed no antitumor effects.

It is significant that the differences in efficacy with more establishedtumors are greater with the higher antigen expressing tumor line, H2707.The observation that H2707 has a greater response to BR96 therapy thanH2987 is consistent with the assumption that the amount of antigenexpressed by a tumor cell may influence the efficacy of BR96 treatment.From the data above it is clear that BR96 has antitumor effects againststaged tumors.

Dose Effects of BR96 Antibody

In another experiment, the dose effects of BR96 against the H2707 tumorline were examined. In this experiment, ER96 was administered indecreasing half log amounts from 1 mg/dose to 0.032 mg/dose. The meantumor volumes versus time post tumor implant of the groups are presentedin FIG. 25. The control treated animals were given only the highest doseof monoclonal antibody, 1 mg/dose FA6. These control animals showed noantitumor effects while there was a dose dependent response when BR96antibody was administered over the chosen dose range.

Antitumor Effects of F(ab')₂ Fragment and Chimeric BR96

In addition, antitumor effects of the F(ab')₂ BR96 fragment wereexamined to determine if the antitumor effects seen in vivo were due tothe Fc portion or if actual binding to the tumor with its subsequentinternalization was sufficient for cell death, as indicated by in vitroassays. The dose of F(ab')₂ fragment was 0.66 mg/dose using the sameschedule as the whole BR96. This dose corresponds to an approximatemolar equivalent of binding regions compared to the 1.0 mg/dose wholeIgG₃ BR96. Mean tumor volume values versus time post tumor implantationfor this group treated with the antibody fragment are shown in FIG. 26.There were clearly some antitumor effects although the effects were notas strong as with whole antibody. These effects were most pronounced atthe earlier time points during and immediately following treatment.

Chimeric BR96 was also examined for antitumor effects in thisexperiment. An intermediate dose of 0.32 mg/dose for the chimericmonoclonal antibody was chosen. The mean tumor volume values for thisgroup of mice is also shown in FIG. 26. Treatment with chimeric antibodyBR96 was more efficacious than a comparable dose of the murine BR96IgG₃. This is further demonstrated in FIG. 27 which shows that 6 of the8 mice treated with chimeric BR96 were free of palpable tumors at theend of treatment.

Examination of the individual tumors depicted in FIG. 27 shows that atcompletion of treatment a clear dose effect was evident by the number ofanimals without tumors after treatment with decreasing amounts of wholeIgG3 BR96 antibody from 1.0 to 0.1 mg/dose. Surprisingly, treatment with0.032 mg/dose resulted in an antitumor effect similar to the 0.32mg/dose. This may reflect that the level of cells killed in the tumorfrom the treatment was very close to the minimum amount necessary forthe tumor to continue to grow.

Three of the eight animals treated with the F(ab')₂ fragment were freeof palpable tumors after treatment. Therefore the Fc portion is notentirely necessary for the antibody to have antitumor effects in vivoalthough it should enhance the tumorcidal properties of BR96,particularly in immunocompetent animals.

The above results demonstrate that unmodified BR96 antibodies areeffective antitumor agents against tumor lines in vivo. Moreover, theBR96 antibodies have an effect on staged or established growing tumors.There is an indication that higher antigen density on the tumor line mayincrease the ability of BR96 to kill these cells. It has been shown thatany of the forms of the monoclonal antibody, i.e., chimeric, murinewhole IgG₃ or F(ab')₂ fragments, are effective as antitumor agents.Earlier treatment and higher doses are preferred.

EXAMPLE 12 Localization and Biodistribution of BR96 Antibodies

Radioiodinated BR96 monoclonal antibodies administered at doses used inthe therapy experiments described above in Example 11 were used todetermine biodistribution characteristics. Specifically, the whole IgG₃BR96, chimeric or F(ab')₂ fragments together with the appropriatecontrol (whole monoclonal antibody FA6, chimeric 96.5 and 96.5 F(ab')₂,respectively) were used to localize in the tumor and various otherorgans.

Prior to the localization experiments, animals were injected with tumorcells as described above in Example 11, for the therapy studies.However, the tumors were allowed to grow in the animals forapproximately 2 weeks. At this time, 100 μg of BR96 antibody or fragmentwas radiolabeled with ¹²⁵ I using 10 μg Iodogen for 10 minutes at roomtemperature as directed by the manufacturer. Control antibody orfragments were labeled with ¹³¹ I using the same method. Theseradioiodinated antibodies were diluted with the appropriate unlabeledantibodies to reach the doses used in the therapy experiments. Both thespecific and nonspecific antibodies were then mixed and administeredtogether, i.v., through the tail vein of the mouse. At selected timesmice were randomly pulled from the group, anesthetized, bled through theorbital plexus and sacrificed. Various tissues were removed, weighed andcounted on a gamma counter capable of differentiating between the tworadioisotopes of iodine. From this data, percent injected dose andpercent injected dose per gram were calculated.

The accumulated data from the 24 post administration time point in thelocalization experiments are summarized in Table 7.

                                      TABLE 7                                     __________________________________________________________________________    Summary of Biodistribution Experiments                                                       % INJECTED DOSE/GRAM                                             DOSE TUMOR 24 HRS. POST ADMINISTRATION                                      ANTIBODY                                                                              (mg)                                                                             CELL                                                                              BLOOD                                                                              TUMOR                                                                              LIVER                                                                             SPLEEN                                                                             KIDNEY                                                                             LUNG                                   __________________________________________________________________________    1)                                                                              BR96-G.sub.3                                                                        1.0                                                                              H2987                                                                             10.2 6.8  2.2 1.9  3.4  4.7                                       FA6 1.0  6.3 2.1 2.1 1.6 2.4 3.2                                             2) BR96-G.sub.3 0.3 H2707 9.0 7.0 1.8 1.6 2.7 3.7                              FA6 0.3  5.9 2.7 2.0 1.8 2.2 2.8                                             3) ChiBR96 0.32 H2707 7.2 8.2 1.4 1.6 2.0 3.5                                  Chi96.5 0.32  7.5 2.3 1.8 1.6 1.9 3.5                                        4) F(ab').sub.2                                                                BR96 0.65 H2707 <0.3 <0.3 <0.3 <0.3 <0.3 <0.3                                 96.5 0.65  <0.3 <0.3 <0.3 <0.3 <0.3 <0.3                                   __________________________________________________________________________

The only tissue showing significant differences between specific andnonspecific antibody is the tumor. All other tissues examined showapproximately equal uptake between the specific and nonspecificantibodies. One possible exception is the lower blood levels for thenonspecific antibody, FA6. This indicates accelerated blood clearance ofthis antibody. However, the difference between the specific andnonspecific antibody in the tumor is greater than the difference inblood levels between the FA6 and BR96 antibodies.

The data in Table 7 also demonstrate that the percent of the dosepresent in a particular organ is constant regardless of the doseadministered. This would therefore indicate there is quantitatively moreantibody present at the tumor site when higher doses are administered.In addition, there are no apparent differences between the two tumorlines with respect to specific vs nonspecific uptake.

Table 7 also demonstrates that the F(ab')₂ was cleared from the animalat a much faster rate than either the IgG₃ or chimeric BR96. This couldexplain the reduction in efficacy of the fragment compared to wholeantibody in the therapeutic experiments. Any antitumor effects from thefragment must therefore be rapid and occur during the short time spanprior to being cleared.

ChiBR96 localized at a comparable level to the IgG₃ BR96. Higher amountswere present only in the tumor compared to the control chimericantibody. This suggests that any increase in efficacy of the chimericantibody compared to the murine BR96 IgG₃ is due to the human constantregion substitution. Of equal importance, the human constant regionsubstitution does not appear to affect the ability of the chimericantibody to localize to the tumor or adversely affect itsbiodistribution.

In summary, the IgG₃ and chimeric forms of BR96 are capable ofspecifically localizing to the tumor site. Moreover, both localizationand therapeutic effects have been shown in these preliminary experimentsat comparable doses. Indirect evidence of localization of the F(ab')₂fragments was shown by the antitumor activity of the fragments in thetherapy experiments. This activity must occur before 24 hours.

EXAMPLE 13 Preparation and Cytotoxicity of Chimeric Monoclonal AntibodyF(ab')₂ and Fab' Fragments Conjugated to Pseudomonas Exotoxin

Cell-specific cytotoxic reagents were prepared by chemically combiningthe chimeric antibody BR96 (ChiBR96) with Pseudomonas exotoxin A (PE)using either native PE or a truncated form (LysPE40) devoid of the cellrecognition region (Domain I). A variety of chimeric BR96-immunotoxinswere constructed by chemical conjugation of PE and LysPE40 with Fab' orF(ab')₂ enzymatic digest products, or BR96 antibody, by thiolation with2-iminothiolane or by direct attachment to intact BR96 antibody byreduction with DTT as described below.

Reagents

Succinimidyl 4-(N-maleimido-methyl) cyclohexane 1-carboxylate (SMCC) and2-iminothiolane (2-IT) were purchased from the Pierce ChemicalCorporation (Rockford, Ill.). Soluble pepsin was purchased from SigmaChemical Co. (St. Louis, Mo.). Na[¹²⁵ I] and [³ H]-leucine werepurchased from New England Nuclear (Boston, Mass.). Native PE waspurchased from Berna Products (Coral Gables, Fla.). Mono Q columns werepurchased from Pharmacia (Uppsala, Sweden). TSK-3000 columns werepurchased from TosoHaas, Inc. (Philadelphia, Pa.). Immunoblots wereperformed using mouse (anti-id BR96) and rabbit (anti-PE) ABC kits(Vector Laboratories, Burlingame, Calif.). Rabbit polyclonal anti-PEantibody and mouse anti-PE monoclonal antibody M40/1 were supplied byDrs. Ira Pastan and David FitzGerald, National Institutes of Health(Bethesda, Md.). Anti-idiotypic BR96 antibody 757-4-1 was prepared usingthe BR96 antibody of the invention and standard procedures for preparinganti-id antibodies [see, Kahn et al., Cancer Res. 49: 3159-3162 (1989)and Hellstrom et al., Cancer Res. 50: 2449-2454 (1990)] by Dr. BruceMixan, Bristol-Myers Squibb (Seattle, Wash.)].

Cell Culture and Plasmids

All cells were cultured in RPMI 1640 supplemented with 10% fetal bovineserum, except L929, which was cultured in DMEM supplemented with 10%fetal bovine serum. Plasmid pMS8 (FIG. 36), which encodes the gene forLysPE40 under control of the T7 promoter, was constructed by Dr. ClaySiegall (provided by Dr. Ira Pastan, NIH, Bethesda, Md.) from the vectorpVC85 [Kondo et al., J. Biol. Chem. 263: 7470-7475 (1988)] by insertingat the amino terminus a lysine residue and also inserting a multiplecloning site.

Expression and Purification of LysPE40

The plasmid pMS8 encoding LysPE40 was transformed into E. coli BL21(λDE3) cells and cells were cultured in Super Broth (Digene, Inc.,Silver Spring, Md.) containing 75 μg of ampicillin per ml at 37° C. Whenabsorbance at 650 was 2.0 or greater, isopropyl1-thio-β-D-galactopyranoside was added (1 mM) and cells were harvested90 minutes later. The bacteria were washed in sucrose buffer (20%sucrose, 30 mM Tris-HCl (pH 7.4), 1 mM EDTA), and osmotically shocked inice-cold H₂ O to isolate the periplasm. LysPE40 protein was purifiedfrom the periplasm by successive anion-exchange and gel-filtrationchromatographies using a Pharmacia fast protein liquid chromatography(FPLC) system as described previously [Batra et al., Proc. Natl. Acad.Sci. USA 86: 8545-8549 (1989) and Siegall et al., Proc. Natl. Acad. Sci.USA 85: 9738-9742 (1988)].

Generation of BR96 F(ab')₂ and Fab' Fragments

F(ab')₂ fragments were generated from ChiBR96 (4 mg/ml) by pepsindigestion (25 μg/ml in 0.1 M citrate buffer, pH 4.0, [Parham, inHandbook of Experimental Immunology, Weir, Ed., Blackwell ScientificPublishers, p. 1-23 (1986)]. After 6 hours incubation at 37° C.,digestion was terminated by adjusting the pH to 7.2 with PBS. Purity ofthe digest preparation was 90-95% F(ab')₂ determined by SDS-PAGE (4-20%gradient gels) and Coomassie blue staining.

Fab' was prepared from the ChiBR96 F(ab')₂ by reduction with cysteine tobreak the remaining interchain disulfide bonds [Parham et al., supra].Briefly, F(ab')₂ molecules (2-4 mg/ml) in 0.1 M Tris-HCl (pH 7.5) wereincubated at 37° C. for 2 hours with cysteine (0.01 M finalconcentration). Free sulfhydryl groups on the Fab' molecule werealkylated with 0.02 M iodoacetamide (CalBiochem, San Diego, Calif.) for30 minutes at room temperature to prevent recombination of the Fab' toF(ab')₂. The reaction mixture was dialyzed against PBS. Purity wasgreater than 85% as assessed by SDS-PAGE.

Immunotoxin Construction and Purification

Chimeric BR96 (6-10 mg/ml) was thiolated by addition of a 3-fold molarexcess of 2-iminothiolane (2-IT) in 0.2 M sodium phosphate (pH 8.0), 1mM EDTA for 1 hour at 37° C. [Batra et al., Proc. Natl. Acad. Sci. USA86: 8545-8549 (1989)], which introduces sulfhydryl groups by reaction of2-iminothiolate with primary amines. Unreacted 2-IT was removed by PD-10column chromatography (Pharmacia). Alternatively, free thiol groups weregenerated by reduction with dithiothreitol (DTT). Chimeric BR96 wasincubated with a 20-fold molar excess of DTT for 2.5 hours at 42° C.Excess DTT was removed by overnight dialysis against PBS under nitrogen.The number of thiol groups on the monoclonal antibody was determined byDTNB reduction (Ellman's reagent, Sigma Chem. Co.) as described byDeakin et al., Biochem. J. 89: 296-304 (1963), incorporated by referenceherein. This procedure routinely gave 4 thiol groups per BR96 antibody,with no reduction in antibody binding reactivity or proteinconcentration. The procedure was not used with F (ab')₂ or Fab'fragments.

Thiolated BR96 antibody was condensed with maleimide-modified PE orLysPE40. A non-cleavable maleimide group was attached to lysine residueson the toxin (PE or LysPE40; 6-8 mg/ml) by mixing with 3-fold molarexcess of SMCC in 0.2 M sodium phosphate (pH 7.0), 1 mM EDTA at roomtemperature for 30 minutes and purified on a PD-10 column. Modifiedtoxin and thiolated antibody were mixed in a 4:1 molar ratio andincubated at room temperature for 14-16 hours to allow a thioetherlinkage to form. Immunotoxins were purified by anion-exchange (Mono Q)to remove unreacted antibody and gel-filtration chromatography(TSK-3000) to remove unconjugated toxin as previously described by Kondoet al., J. Biol. Chem. 263: 9470-9475 (1988); and Batra et al., Proc.Natl. Acad. Sci. USA 86: 8545-8549 (1989), all incorporated by referenceherein.

Chimeric BR96 IgG-LysPE40 (190 kDa), Fab'-LysPE40 (96 kDa) and F(ab')₂-LysPE40 (145 kDa) conjugates were additionally analyzed by non-reducingSDS-polyacrylamide gel electrophoresis (SDS-PAGE) to determine the sizeof the native conjugate (FIG. 28). From the Coomassie blue stained gels,it was determined that there was less than 5% unconjugated antibodyafter purification.

Binding Studies

For competition binding studies, L2987 cells (Bristol-Myers Squibb Co.,Seattle, Wash.) were removed from monolayer culture using 0.2% trypsinand washed with RPMI 1640 containing 2% FCS (wash buffer). Cellsuspensions (1.0×10⁶ cells/0.1 ml) were incubated with 0.1 mlfluorescein isothiocyanate (FITC)-labeled ChiBR96 (13.3 μg/ml finalconcentration) and 0.1 ml of diluted antibody or immunotoxin at 4° C.for 1 hour, washed, and the amount of cell-bound FITC labeled-ChiBR96was quantified on an EPICS V model 753 Flow Cytometer (Coulter Corp.,Hialeah, Fla.).

For direct binding studies, two-fold serially diluted immunotoxins orantibody was incubated for 1 hour at 4° C. in 0.2 ml wash buffercontaining 1×10⁶ L2987 cells. Cells were washed and then incubated inwash buffer containing 1:40 diluted FITC labelled goat anti-human kappaantibody (Bethyl Labs, Montgomery, Tex.) for an additional 30 min at 4°C. to quantitate cell-bound antibody. Cells were washed and analyzed forcell surface fluorescence on a flow cytometer to determine the amount ofimmunotoxin or antibody remaining on the cell surface.

Two methods were used to determine whether there was an alteration inantibody binding activity after conjugation to PE or LysPE40.Competition binding analysis showed that both immunotoxins competed withFITC-labeled ChiBR96 as efficiently as unconjugated ChiBR96 antibody(FIG. 29), indicating that binding affinity for the BR96 antigen was notperturbed after chemical conjugation. Similar results were obtainedusing the direct binding assay for both PE and LysPE40 conjugates.

Binding activities of LysPE40 conjugated and unconjugated IgG, F(ab')₂and Fab' were also compared by direct binding to L2987 tumor cells.Cell-bound antibody protein was quantitated using FITC-labeled goatanti-human kappa light chain antibody. Binding of the LysPE40immunotoxin was similar to that obtained using unconjugated ChiBR96antibody (FIG. 30A) and agreed with results obtained using thecompetition binding assay (FIG. 29). FIG. 30B compares the bindingactivity of intact IgG to F(ab')₂ and F(ab')₂ -LysPE40. There was noloss in immunoreactivity with the F(ab')₂ and F(ab')₂ -immunotoxin ascompared to ChiBR96 IgG. Conjugation of PE40 to Fab' did not affectimmunoreactivity (FIG. 30C), however, binding of the Fab' wassignificantly decreased as compared to intact IgG (FIG. 30C), mostlikely because of the monovalency of the Fab' molecule.

Antigenic Modulation and Internalization

Modulation of intact ChiBR96, F(ab')₂ or Fab' immunotoxins was assayedon L2987 cells propagated as 90-95% confluent monolayer cultures in 96well microtiter plates as described above. Target cells were pulsed for1 hour at 4° C. with 0.1 ml of two-fold serially diluted immunotoxinranging from 5-800×10⁻⁷ M antibody protein in binding buffer. Monolayercultures were washed free from unbound material using growth medium andindividual plates were incubated in complete medium under eithernon-modulating (4° C.) or modulating (37° C.) conditions.

The amount of membrane-associated immunotoxin bound to target cellpopulations at each time point shown in FIG. 31 was quantified using[¹²⁵ I]M40/1 (anti-PE) antibody (provided by Dr. D. Fitzgerald, NCI,Bethesda, Md., [Ogata et al., Inf. and Immun. 59: 407-414 (1991)]).Epitope mapping of M40/1 antibody determined that it binds to a 44 aminoacid region in the PE domain II [Ogata et al., supra]. Monoclonalantibody M40/1 was radioiodinated using Na[¹²⁵ I] (New England Nuclear,Boston, Mass.) and chloramine T (Kodak Chemical Co., Rochester, N.Y.) asdescribed by McConahey and Dixon, Arch. Allergy Appl. 29: 185-188(1966), incorporated by reference herein. Radioiodinated M40/1 wasseparated from unbound iodine by PD10 column chromatography (Pharmacia).Specific activities ranged from 2 to 5×10⁵ CPM/μg.

At various times during incubation at 37° C. or 4° C., a triplicate setof wells were twice washed with wash buffer and pulse-labeled with 0.1ml [¹²⁵ I]-labeled M40/1 antibody (0.5 μg/ml in wash buffer containing0.2% sodium azide) to determine membrane bound conjugate. After 15minutes, monolayers were washed free of unbound label, and cell-boundcpm was determined by solubilization of the cell monolayer with 0.5 NNaOH. Cell-bound radioactivity was determined using a LKB model 1272gamma counter. Non-specific binding was determined by incubation oftarget cells with a similar concentration of unconjugated ChiBR96. Incertain experiments, unconjugated PE was used to determine backgroundbinding levels. [¹²⁵ ]-labeled M40/1 antibody did not react withmembrane bound antibody or PE.

The ability of ChiBR96-PE and ChiBR96-LysPE40 to induce antigenicmodulation was initially measured by determining the loss of immunotoxinfrom the cell surface membrane (FIG. 31). There was no difference inmodulation kinetics between PE or LysPE40 immunotoxins withapproximately 50% of the original cell-bound immunotoxin modulated fromthe surface membrane 30 minutes after warming to 37° C. Cells incubatedunder conditions which do not allow antigenic modulation (4° C.), showedessentially no loss of cell surface toxin within 6 hours (FIG. 31A).

In order to confirm that the loss of cell-surface immunotoxin was due toendocytosis, cells were incubated with a [¹²⁵ I]-labeled immunotoxincomplex for 1 hour at 4° C. to permit binding, washed and weresubsequently modulated at 37° C. As shown in FIG. 31B, essentially allthe radiolabeled immunotoxin remained cell-associated, despite theconcomitant loss from the cell-surface membrane (FIG. 31A). Thesefindings confirm that most if not all of the membrane-associated BR96immunotoxins were rapidly internalized, and that internalization rateswere similar for PE and LysPE40 immunotoxins.

The capacity of ChiBR96 F(ab')₂ -LysPE40 and Fab'-LysPE40 immunotoxinsto internalize was also determined by measuring the loss of cell-surfaceimmunotoxin using radiolabeled anti-PE antibody. Essentially all of theChiBR96 immunotoxins were completely internalized after 4.5 hoursincluding the Fab'-immunotoxin (Table 8). However, rate differences wereobserved. At 2.5 hours, when 76% of the intact IgG toxin and 72% of theF(ab')₂ were internalized, only 12% of the Fab' immunotoxin wasinternalized. Therefore, both IgG, F(ab')₂ and Fab'-LysPE40 immunotoxinswere modulated from the cell surface membrane, but at different rates.

                  TABLE 8                                                         ______________________________________                                        Internalization of BR-96-Immunotoxins                                           from the Cell Surface Membrane of L2987 Cells                                              % INTERNALIZATION                                                           2.5 hr  4.5 hr                                                   ______________________________________                                        BR-96-LysPE40  74.0      85.0                                                   F9(ab').sub.2 -LysPE40 72.0 91.6                                              Fab'-PE40 12.0 89.6                                                         ______________________________________                                    

Inhibition of Protein Synthesis Assay

Cytotoxicity of various forms of ChiBR96 antibody conjugated to LysPE40against tumor cells was determined by measuring inhibition of proteinsynthesis as follows: Tumor cells (1×10⁵ cells/ml) in growth media wereadded to 96 well flat bottom tissue culture plates (0.1 ml/well) andincubated at 37° C. for 16 hours. Dilutions of toxin or toxin-conjugateswere made in growth media and 0.1 ml added to each well (3wells/dilution) for 1 hour or 20 hours at 37° C. After the appropriateincubation time, unreacted material was removed by washing the monolayerwith growth media. Cells were incubated in 0.2 ml growth media for atotal of 20 hours and pulse-labeled with [³ H]-leucine (1 μCi/well) foran additional 4 hours at 37° C. The cells were lysed by freezing,thawing at 37° C. and harvested using a Tomtec cell harvester (Orange,Conn.). Cellular protein labeled with [³ H]-leucine was determined bycounting the radioisotope using a LKB Beta Plate (LKB, Piscataway, N.J.)liquid scintillation counter.

Analysis of Competition of Immunotoxin Cytotoxic Activity

Chimeric BR96-PE40 was added at 0.8, 4 and 20 pM concentrations to MCF-7cells in the presence or absence of 50 μg (333 pM) ChiBR96 antibody.Cytotoxicity was determined as described in the inhibition of proteinsynthesis assay as described above.

In vitro Cytotoxicity of Intact IgG-PE Immunotoxins

The in vitro cytotoxic activity of the immunotoxins against cancer cellswas assayed by comparing inhibition of protein synthesis on antigenpositive and antigen negative cells (Table 9). BR96 antigen-positivecell lines MCF-7, L2987, and RCA were the most sensitive to ChiBR96-PEwith EC₅₀ values of 0.14, 0.28, and 1.4 pM, respectively. Theimmunotoxin was also more inhibitory than native PE which had EC₅₀values of 200, 140 and 380 pM. When tested on antigen-negative celllines, little difference in EC₅₀ values between PE and the immunotoxinwas observed. Specificity, (antibody-directed cell-killing), must takeinto account the different sensitivities of the various cell lines tonative PE. Thus, the ChiBR96 immunotoxins were 100-500 fold more potentthan native PE against antigen-positive cell lines.

                  TABLE 9                                                         ______________________________________                                        Cytotoxicity of ChiBR96-PE on Human Tumor Cells                                               EC.sub.50, pM                                                                         DTT     2-IT-                                           Cell  BR96 Reduced Treated Native                                             Line Type Antigen ChiBR96-PE ChiBR96-PE PE                                  ______________________________________                                        MCF-7.sup.1                                                                          Breast   +       0.10    0.14    200.0                                   L2987 Lung Ca. + 0.25 0.28 140.0                                              RCA Colon + 1.2 1.4 380.0                                                      Ca.                                                                          A2780 Ovarian - 23.0 23.0 60.0                                                 Ca.                                                                          L929 Mouse - 13.5 14.0 3.0                                                     Fblst                                                                        KB.sup.2 Epider- - 220.0 231.0 227.0                                           moid                                                                       ______________________________________                                         .sup.1 ATCC No. HTB 22                                                        .sup.2 ATCC No. CCL 17                                                        EC.sub.50 represents the amount of immunotoxin or toxin required to           inhibit 50% of the protein synthesis as determined by [.sup.3 Hleucine        incorporation in cellular protein. (BR96 antigen + = Epitope density of >     1 × 10.sup.4 molecules/cell).                                      

Cytotoxicity of ChiBR96 Mab and Enzymatic Fragments Linked to LysPE40Against MCF-7 Cells

Smaller immunotoxin molecules may be beneficial in tumor penetration,therefore, the cytotoxic activity of ChiBR96 as Fab', F(ab')2 fragmentsand as an IgG linked to LysPE40 was compared (Table 10). As with theChiBR96-PE immunotoxin (Table 9), MCF-7 and L2987 cells were the mostsensitive cell lines tested. The IgG and F(ab')₂ -LysPE40 moleculesshowed similar cytotoxic activity against MCF-7 cells (EC₅₀ =8-14 pM)while the Fab'-LysPE40 conjugate was much less active (EC₅₀ =780 pM)(FIG. 32). Specificity of protein synthesis inhibition activity of Fab'and F(ab')₂ conjugates was also preserved, with little or no inhibitoryactivity observed against the antigen-negative cell lines A2780.

                  TABLE 10                                                        ______________________________________                                        Cytotoxicity of 2-iminothiolane Substituted                                     Chimeric BR96-PE40 on Human Tumor Cells                                                        BR96 EC.sub.50, pM                                         Cell           Anti-  BR96   F(ab').sub.2                                                                        Fab'-                                        Line Type gen PE40                                                           PE40 PE40 PE40                                                               ______________________________________                                        MCF-7 Breast   ++      8     14     780   15,000                                 Ca.                                                                          L2987 Lung + 37 70 2700 17,500                                                 Ca.                                                                          RCA Colon + 84 110  5000 15,000                                                Ca.                                                                          A2780 Ovarian - 650  2500  11,000   15,000                                     Ca.                                                                          KB Epider- - >5000    N.D. N.D. >25,000                                        moid Ca                                                                    ______________________________________                                         EC.sub.50 is described in Table 8 legend. N.D. = not determined.         

Specificity of Growth Inhibition By ChiBR96-LysPE40

Specificity was confirmed by abrogating the protein synthesis inhibitionby ChiBR96-LysPE40 with unconjugated ChiBR96. Addition of excess ChiBR96antibody (50 μg) with ChiBR96-LysPE40 immunotoxin, resulted in adecrease of in vitro potency (FIG. 33). At 20 pM of ChiBR96-LysPE40,approximately 50% of its cytotoxic effect was blocked by the addition ofexcess unconjugated antibody, while at 4 pM, the excess ChiBR96completely blocked the cytotoxic activity of ChiBR96-LysPE40.

Kinetics of Cytotoxicity of ChiBR96 Immunotoxins and Native PE

In part, the effectiveness of immunotoxins may depend on the rate ofinternalization after binding to antigen-positive cells. To determinethe cytotoxic activity of ChiBR96-PE, ChiBR96-LysPE40 and PE, a timecourse analysis was performed where cells were incubated with toxin forup to 20 hours as described above.

After 1 hour incubation, MCF-7 cells were sensitive to ChiBR96-PE andChiBR96-LysPE40 (EC₅₀ values of 1 and 60 pm, respectively) but not tothe native toxin (EC₅₀ >10,000 pM). After 20 hours MCF-7 cells wereslightly more sensitive to ChiBR96-PE and ChiBR96-PE40, but much moresensitive to PE; EC₅₀ =200 pM (FIG. 34). This assay was repeated at 2,4, and 6 hour time points. At each time point, PE was considerably lesscytotoxic against MCF-7 cells than ChiBR96-immunotoxins. This may be duein part to the mechanism by which the toxin molecule is delivered totarget cells.

This example demonstrates the production of immunotoxins containing thecarcinoma-associated monoclonal antibody ChiBR96 and Pseudomonasexotoxin A. The antibody was used in forms including native IgG, reducedIgG, F(ab')₂ and Fab'. The toxin component of the immunotoxin was eithernative PE or LysPE40, a truncated form containing a genetically modifiedamino terminus that includes a lysine residue for conjugation purposes.Chimeric BR96-toxin conjugates were found to be cytotoxic to cells whichdisplay Lewis Y, a determinant recognized by the BR96 monoclonalantibody of the invention. The most cytotoxic of the conjugates producedwas ChiBR96-PE which was 1000-fold more potent than PE itself againstMCF-7 breast carcinoma cells. Chimeric BR96-LysPE40 was also extremelycytotoxic towards BR96 antigen positive cells (1000-fold more potentthan LysPE40). Both ChiBR96-PE and ChiBR96-LysPE40 were produced usingtwo procedures which generated sulfhydryl groups on the antibody, bymild reduction of the antibody or by derivatizing the antibody with2-iminothiolane. The former procedure produced a greater yield ofconjugate, but conjugates produced by both procedures resulted inchimeric molecules of identical activities.

Chimeric BR96-PE and ChiBR96-LysPE40 were almost fully active with 1hour incubation, while PE was relatively inactive (FIG. 34). Withcontinued incubation, ChiBR96-immunotoxins increase cytotoxic activityonly slightly while PE becomes cytotoxic to the MCF-7 cells at latertime points. This rapid efficacy of ChiBR96-immunotoxins is evidence ofthe utility of ChiBR96 in targeting cell populations for killing.

The binding and internalization activities of ChiBR96-immunotoxins werealso examined. Immunoconjugates prepared with intact IgG or its F(ab')₂or Fab' enzymatic digest products were not affected in terms of bindingby chemical conjugation to LysPE40 (FIG. 30) or PE. Differences inbinding activity between Fab' and F(ab')₂ or IgG conjugates may beattributed to differences in avidity due to the monovalence of the Fab'molecule. We also cannot exclude the possibility that enzymaticdigestion used to generate the Fab' fragments contributed to thedecreased avidity. Of most interest was the comparison betweenChiBR96-LysPE40 and the enzymatic fragment immunotoxins ChiBR96 F(ab')₂-LysPE40 and ChiBR96 Fab'-LysPE40. This finding is also reflected in thecytotoxicity data (Table 10).

The results presented in this example demonstrate that both intact PEand LysPE40 immunotoxins as well as F(ab')₂ and Fab' immunotoxinsdemonstrate cytotoxic activity in vitro.

EXAMPLE 14 Preparation of Single-Chain BR96 sFv-PE40 Immunotoxin

This example describes the preparation and characterization ofcytotoxicity of a single-chain immunotoxin, BR96 sFV-PE40, consisting ofthe cloned heavy and light chain Fv portions of the BR96 monoclonalantibody of the invention linked to PE40. Q Sepharose was purchased fromPharmacia (Uppsala, Sweden). TSK-3000 columns were purchased fromTosoHaas, Inc. (Philadelphia, Pa.). Immunoblots were performed usingmouse anti-idiotypic BR96 antibody 757-4-1 as described above in Example13, and ABC immunoblot kits (Vector Laboratories, Burlingame, Calif.).Chloramine T was purchased from Sigma Chemical Co. (St. Louis, Mo.).MCF-7 human breast carcinoma cells were originally obtained from theATCC (Rockville, Md.) and have been maintained by Bristol-Myers SquibbCompany, Seattle, Wash. RCA colon carcinoma cells were obtained from M.Brattain, Baylor University, Texas. L2987 lung adenocarcinoma cells wereobtained from Dr. I. Hellstrom, Bristol-Myers Squibb Co., Seattle, Wash.A2780 ovarian carcinoma cells were obtained from K. Scanlon, NIH,Bethesda, Md., and KB epidermoid carcinoma cells were obtained from Dr.Ira Pastan, NIH, Bethesda, Md. Cells were cultured in RPMI 1640supplemented with 10% fetal bovine serum.

Construction of BR96 sFv-PE40

In order to produce a single-chain recombinant immunotoxin, the Fvdomains of the light and heavy chains of BR96 IgG were isolated fromplasmid pBR96 Fv (FIG. 36) containing the BR96 Fv sequences using PCRamplification.

Identification of Primers for PCR Amplification

Two sets of PCR primers:

Primer 1: 5'-GCTAGACATATGGAGGTGCAGCTGGTGGAGTCT-3' (SEQ ID NO: 1) andprimer 2: 5'-GCTGTGGAGACTGGCCTGGTTTCTGCAGGTACC-3' (SEQ ID NO:2) weredevised for the amplification of the V_(L) and V_(H) of murine chimericBR96 monoclonal antibody. The V_(L) and V_(H) -5' PCR primers were basedon the N-terminal amino acid sequence of the BR96 light and heavy chains(FIG. 35, SEQ ID NO:3), respectively, while the 3' primers were designedaccording to the frequency of the most common nucleotide at eachposition of joining (J)-region segments after alignment of V_(H) andV_(k) genes [Kabat et al., in Sequences of Proteins Of ImmunologicalInterest, U.S. Dept. Health and Human Services, Washington, D.C.(1987)]. The V_(L) -5' primer was comprised of 24 nucleotides encodingthe N-terminal amino acids of the variable light chain and a Hind IIIsite 5' to these nucleotides while the V_(L) -3' primer consisted of 22nucleotides which were complementary to the J region of mouse kappalight chain mRNA and a Sph I site 5' to these nucleotides. The V_(H) -5'primer contained 30 nucleotides encoding the N-terminal amino acids ofthe heavy chain and an Eco RI site 5' to these nucleotides while theV_(H) -3' primer contained 22 nucleotides with J region complimentarilyand a BamHI site 5' to these nucleotides. In designing each primer,additional nucleotides were incorporated at the 5' end in order tooptimize restriction site digestion and subsequent cloning of the PCRreaction products. The 5' PCR primer (primer 1) was designed to encode aunique Nde I restriction site.

RNA Isolation, cDNA Synthesis and Amplification

RNA was prepared from about 1×10⁸ BR96 hybridoma cells grown in IMDMsupplemented with 10% fetal calf serum (FCS). Total RNA was used forfirst strand cDNA synthesis using random hexamers at 23° C. for 10minutes in a 20 μl reaction mixture containing 1 μg of total RNA, 4 mMMgCl₂, 1 mM of each dNTP, 1 unit of RNAase inhibitor (recombinant RNAaseinhibitor originally isolated from human placenta, Perkin-Elmer/Cetus,Norwalk, Conn.), 1×PCR buffer (10×PCR Reaction buffer=500 nM KCl, 100 mMTris-HCl, pH 8.3), 2.5 μM random hexanucleotide (Perkin-Elmer/Cetus) and2.5 units of reverse transcriptase (cloned Moloney murine leukemia virus[M-MLV] reverse transcriptase, 2.5 units/μl from Perkin-Elmer/Cetus).Subsequent to hexameric primer extension with reverse transcriptase, thereaction mixtures were incubated successively in a thermal cycler(Eppendorf MicroCycler) at 42° C. for 15 minutes, 99° C. for 5 minutesand 5° C. for 5 minutes. Amplification of V_(L) and V_(H) cDNAs wasperformed with 35 cycles of PCR using reagents according tomanufacturer's instructions (GeneAmp RNA-PCR, Perkin-Elmer/Cetus) in twoseparate tubes with 0.15 μM each of either V_(L) -5' and V_(L) -3 orV_(H) -5' and V_(H) -3' primers. Each PCR cycle consisted ofdenaturation at 95° C. for 1 minute followed by annealing and extensionat 60° C. for 1 minute. In order to fully extend all cDNAs, a singleheld extension was performed at 60° C. for 7 minutes.

Cloning of Amplified cDNA

The amplified PCR products were purified on ion-exchange mini-columns(Elutip-D, Schleicher & Schuell, Keene, N.H.), concentrated by ethanolprecipitation and digested with either EcoRI and BamHI (V_(H) gene) orHind III and Sph I (V_(L) gene). Subsequently, the digested PCR reactionproducts were further purified on 1.5% agarose gels (SeaKem, FMC Corp.Rockland, Me.) and the V_(H) or V_(L) gene fragments separately clonedinto pEV3-SM2 [Crowl et al., Gene 38: 31-38 (1985)] digested with eitherEcoRI and BamHI or with Hind III and Sph I respectively. Clonescontaining V gene inserts were identified by colony hybridization usingeither random-primed radiolabeled V_(L) or V_(H) cDNA fragments asprobes. The nucleotide sequence was then determined for several clonedV_(L) or V_(H) cDNA inserts, employing primers based upstream within thelambda P_(L) promoter or downstream of Sal I within pBR322 sequences.

Construction of Plasmid pBW 7.0

Starting with the BR96 sFv sequence encoded by plasmid pBR96Fv (FIG. 35,prepared by Dr. McAndrew, Bristol-Myers Squibb Co.) a 550 bp sequencecorresponding to the variable heavy and variable light chains connectedwith a synthetic (Gly₄ Ser)₃ hinge region up to the Kpn I restrictionsite in the light chain, was used to PCR-amplify with primer 1 andprimer 2 described above. After PCR-amplification and digestion with NdeI and Kpn I the 550 bp Nde I-Kpn I fragment was ligated into a 4220 bpNde I-Kpn I vector fragment prepared from plasmid pMS8 described above,(supplied by Dr. Ira Pastan, NIH, Bethesda, Md.), which encodes the genefor PE40 under the transcriptional control of the T7 promoter [Studieret al., J. Mol. Biol. 189: 113-130 (1986)]. The product of this ligationwas an intermediate vector designated pBW 7.01 (FIG. 35). Subsequently,the 227 bp Kpn I fragment from pBR96 Fv was subcloned into the uniqueKpn I site of pBW 7.01. The resulting plasmid pBW 7.0 (FIG. 36),encoding the BR96 sFv-PE40 gene fusion, was confirmed by DNA sequenceanalysis.

Expression and Purification of BR96 sFv-PE40

The plasmid pBW 7.0 encoding BR96 sFv-PE40 obtained as described abovewas transformed into E. coli BL21 (λ DE3) cells cultured in Super Broth(Digene, Inc., Silver Springs, Md.) containing 75 μg of ampicillin perml at 37° C. When absorbance at 650 nm reached 1.0, isopropyl1-thiol-B-D-galactopyranoside (IPTG) was added to a final concentrationof 1 mM, and cells were harvested 90 minutes later. Upon induction withIPTG, the E. coli cells transformed with pBW 7.0 expressed large amountsof fusion protein that was localized to the inclusion bodies. Thebacteria were washed in sucrose buffer (20% sucrose, 30 mM Tris-HCl (pH7.4), 1 mM EDTA) and were osmotically shocked in ice-cold H₂ O toisolate the periplasm. Subsequently, inclusion bodies were isolated awayfrom the spheroplast membrane proteins by extensive treatment withTergitol (Sigma) to remove excess bacterial proteins, followed bydenaturation in 7 M guanidine-HCl (pH 7.4), refolding in PBSsupplemented with 0.4 M L-Arginine and extensive dialysis against 0.02 MTris, pH 7.4. Protein was purified using anion-exchange on a Q-Sepharosecolumn and fractions containing BR96 sFv-PE40 were then pooled andseparated by gel-filtration (on a TSK-3000 column) chromatographies witha Pharmacia fast protein liquid chromatograph (FLPC) system as describedby Siegall et al., Proc. Natl. Acad. Sci. USA 85: 9738-9742 (1988).

The chromatographic profile of the size exclusion column indicated thepresence of two major species (FIG. 37A). The first species elutedbetween gel filtration standards of 660 kD and 158 kD and represents anaggregated form of the recombinant protein (fractions 9-14). The secondspecies (fractions 15-21) represents the 67 kDa monomeric form of BR96sFv-PE40 which, as expected, eluted between the 158 kDa and 44 kDastandards. These results were confirmed by reducing (FIG. 37B) andnon-reducing SDS-PAGE analysis (FIG. 37C). Whereas FIG. 37B shows thepurification profile based on Coomassie staining, FIG. 37C showsimmunoblot analysis using anti-idiotypic BR96 antibody. The above datademonstrate that purification yielded two forms of recombinant protein,monomers and aggregates.

Direct Lewis Y Determinant Binding ELISA

Because BR96 sFv-PE40 is monovalent it provides only one antigen-bindingsite per molecule. In order to test the relative binding activities ofmonovalent BR96 sFv-PE40 compared to the bivalent BR96 antibody, adirect binding assay was performed in which purified Lewis Y was coatedon ELISA plates and the recombinant BR96 sFv-PE40 molecule was compared,in its ability to bind to the antigen, with several antibodies andantibody fragments. Lewis-Y (ChemBiomed, Alberta, Canada) was diluted to0.2 μg/ml in Coating Buffer (100 mM sodium carbonate/bicarbonate, pH9.4) prior to coating Dynatech Immunon II plates and incubating for 16hours at 4° C. Excess antigen was removed and the plates were blockedwith PTB buffer (PBS containing 0.05% Tween 20 and 1% BSA) for 1 hour atroom temperature followed by 3 washes with PTB. The antibody sampleswere serially diluted in PTB to a final concentration ranging from 1.25μg/ml to 80 μg/ml and incubated overnight at 4° C. on the plate in avolume of 50 μl/well. The plates were washed 3 times with PTB buffer andeach well was incubated with 100 μl/well of biotinylated BR96anti-idiotypic antibodies (2.56 μg/ml) in PTB for 1 hour at roomtemperature. The plates were then washed 4 additional times with PTB.Alkaline phosphatase-conjugated streptavidin (Kirkegaard & Perry Labs,Gaithersburg, Md.) was added to each well (100 μl of 0.5 μg/ml in PBScontaining 1% BSA) and incubated for 1 hour at 37° C. Plates were washed3 times with PTB and 3 times with phosphatase buffer (75 M Tris, 0.1 MNaCl, 5 mM MgCl₂, pH 9.4) and reacted with p-nitrophenyl phosphate (1 mMin phosphatase buffer) for 30 to 60 minutes at 37° C. The reaction wasstopped by the addition of 2 N NaOH. The plates were read at 405 nm on aMolecular Device, Inc. (Menlo Park, Calif.) microplate Reader.

In comparison with BR96 IgG, monomeric BR96 sFv-PE40 bound approximately5-fold less well (FIG. 38). In contrast, the aggregated form of BR96sFv-PE40 was unable to bind to the Lewis-Y determinant. L6 IgG, anantibody that does not bind the BR96 antigen, was used as a negativecontrol.

In addition, the competitive binding ability of BR96 sFv-PE40 wascompared with BR96 IgG. Microtiter plates were coated with Lewis-Yantigen as described above. Antibody samples were diluted in PBScontaining 1% BSA to final concentrations ranging from 1.36 μg/ml to 175μg/ml. ¹²⁵ I-BR96 IgG was added to each sample (5 μCi/ml) along withantibody competitor to a final volume of 100 μl. The entire mixture ofradiolabeled BR96 IgG and antibody competitor were added to the Lewis-Ycoated plates and incubated for 2 hours at 37° C. The plates were washedfive times with PBS containing 0.05% Tween-20 and the wells counted on agamma counter. This assay, which also used Lewis-Y coated plates,measured the amount of bound radioiodinated BR96 IgG when compared withvarious amounts of BR96 sFv-PE40 or BR96 IgG.

The results of the competitive binding assay were that BR96 sFv-PE40competed 5-fold less well than BR96 IgG (FIG. 39) which correlates withthe direct binding data in FIG. 38. The addition of L6 IgG, which didnot compete for binding, demonstrates the specificity of this assay.

Cytotoxicity of BR96 sFv-PE40 Against Cancer Cells

To determine the cytotoxic potential of monomeric BR96 sFv-PE40 theeffect of the single-chain immunotoxin was compared to that of thechemical conjugate, ChiBR96-LysPE40 on MCF-7 breast carcinoma cellsmeasured as inhibition of protein synthesis (FIG. 40). Determination ofinhibition of protein synthesis was as follows:

All cell lines were cultured as monolayers at 37° C. in RPMI 1640supplemented with 10% fetal bovine serum, 2 mM L-glutamine and 50units/ml penicillin/streptomycin. Tumor cells were plated onto 96-wellflat bottom tissue culture plates (1×10⁴ cells/well) and kept at 37° C.for 16 hours. Dilutions of immunotoxin were made in growth media and 0.1ml added to each well for 20 hours at 37° C. Each dilution was done intriplicate. The cells were pulsed with [³ H]-leucine (1 μCi/well) for anadditional 4 hours at 37° C. The cells were lysed by freeze-thawing andharvested using a Tomtec cell harvester (Orange, Conn.). Incorporationof [³ H]-leucine was determined by a LKB Beta-Plate liquid scintillationcounter.

For competition experiments, tumor cells were prepared as describedabove. BR96 IgG or, as a control, L6 IgG was diluted to 100 μg/ml ingrowth media before addition to the cell monolayer (0.1 ml/well) . Afterincubation at 37° C. for 1 hour, dilutions of BR96 sFv-PE40 were added,incubated an additional hour, cell supernatants were removed, and cellswere washed with complete RPMI growth media. Growth media (0.2 ml) wasadded to each well, cells were incubated at 37° C. for 20 hours and werelabelled with [³ H]-leucine as described above. The results indicatethat the single-chain immunotoxin was 3-fold more potent than theconjugate, with ID₅₀ values of 4 and 12 pM, respectively.

Next, in order to correlate the cytotoxicity with the presence of theBR96 antigen, the relative antigen density was determined on five tumorcell lines by FACS analysis (FIG. 41). Assays were performed byfluorescence as described by Hellstrom et al., Cancer Res. 50: 2183-2190(1990). Briefly, target cells were harvested in logarithmic phase withEDTA (0.02%) in calcium- and magnesium-free PBS. The cells were washedtwice in PBS containing 1% BSA and resuspended to 1×10⁷ cells/ml in PBScontaining 1% BSA and 0.02% NaN₃. Cells (0.1 ml) were mixed with BR96 ora human IgG control (0.2 ml at 50 μg/ml) and incubated for 45 minutes at4° C. The cells were washed 2 times and resuspended in 0.1 ml of anappropriate concentration of FITC labelled rabbit anti-human IgG(Cappel, Malvern, Pa.). Cells were incubated for 30 minutes at 4° C.,washed 2 times in PBS containing 0.02% NaN₃ and analyzed on a CoulterEPICS 753 fluorescence-activated cell sorter. Data are expressed as thefluorescence intensity of cells reacted with BR96 minus cells reactedwith control antibody. On a logarithmic scale, 25 units of fluorescenceintensity represents a doubling of antigen density. FACS analysis of anon-specific human IgG antibody was performed for each cell line todetermine non-specific fluorescence and a fluorescence intensity wascalculated (Table 11).

                  TABLE 11                                                        ______________________________________                                        Cytotoxicity of BR96 sFv-PE40 on Various Cell Lines                                                  BR96                                                      Cancer Fluorescence ID.sub.50                                                Cell Line Type Intensity ng/ml (pM)                                         ______________________________________                                        MCF-7   Breast     177.8     0.3        (4.4)                                   L2987 Lung 172.8 5.0   (75)                                                   RCA Colon 138.5 8.0   (119)                                                   A2780 Ovarian 103.2 50.0   (750)                                              KB Epidermoid 33.6 500.0 (7,462)                                            ______________________________________                                         ID.sub.50 is the amount of BR96 sFvPE40 required to inhibit 50% of protei     synthesis as determined by [.sup.3 Hleucine incorporation.                    BR96 Fluorescence intensity is the specific BR96 fluorescence intensity       minus nonspecific human IgG fluorescence.                                

When the cytotoxic potential of BR96 sFv-PE40 was tested on the celllines, it was found that inhibition of protein synthesis correlated withBR96 antigen density (Table 11). For example, MCF-7 cells were the mostsensitive to BR96 sFv-PE40 (ID₅₀ of 4.4 pM) of the cell lines tested. Incontrast, KB cells which display negligible amounts of the BR96 antigenwere much less sensitive to BR96 sFv-PE40 (ID₅₀ of 7,462 pM). Thecytotoxic activity of monomeric and aggregated BR96 sFv-PE40 was alsocompared, and the monomer was demonstrated to be approximately 50-60times more effective at inhibiting protein synthesis than the aggregatepopulation with ID₅₀ values on L2987 cells of 75 pM and 2920 pM,respectively.

The competitive cytotoxicity experiments were conducted to confirm thespecificity of the immunotoxin for its antigen binding site (FIG. 42).The cytotoxic effect of BR96 sFv-PE40 is due to specific antigenbinding, because the effect is severely reduced by excess BR96 IgG, butnot by L6 IgG, which does not recognize the BR96 antigen.

Comparative Blood-Level Lifetime Analysis of BR96-Immunotoxins

BR96 sFv-PE40 is approximately one-third the size of the immunotoxinconjugate, ChiBR96-LysPE40. Because protein size can affect biologicalkinetics, the difference in blood half-life between BR96 sFv-PE40 andChiBR96-LysPE40 was measured. Both immunotoxins were radioiodinated andadministered to athymic mice via their tail vein using the followingprocedures.

BR96 sFv-PE40 and ChiBR96-LysPE40 were labelled with Na ¹²⁵ I usingChloramine T [McConahey et al., Arch. Allergy Appl. 29: 185-188 (1966)].Each reaction contained 100 μg of immunotoxin in PBS, 1 μCi of Na¹²⁵ I,and 10 ng/ml chloramine T in a total reaction volume of 100 μl.

After a five minute incubation at room temperature, the reaction wasterminated by addition of 20 ng/ml Na-metabisulfide. The free Na¹²⁵ Iwas separated from the radiolabeled immunotoxin by gel filtrationthrough PD-10 columns (Pharmacia). The specific activity of bothimmunotoxins was approximately 10 μCi/μg.

Female athymic mice (nu/nu) were purchased from Harlan Sprague Dawley(Indianapolis, Ind.) at 4-6 weeks of age. The animals were intravenouslyinjected via the tail vein with 10 μCi of ¹²⁵ I-BR96 sFv-PE40 or ¹²⁵I-ChiBR96-LysPE40. The animal (2-4/data point) were sacrificed atvarious time points and the blood was collected and counted in a gammacounter. The percent (%) ID for the blood was determined as (CPMdetected/CPM injected)×100. ID/ml was calculated assuming a 1.6 ml totalblood volume. Results are shown in Table 12.

                  TABLE 12                                                        ______________________________________                                        Single-Chain Immunotoxin vs. Chemical Conjugate Immunotoxin                     Comparative Blood Level Analysis                                                               BR96     ChiBR96-                                             sFv-PE40 LysPE40                                                              % ID/ml % ID/ml                                                              Time Blood Blood                                                            ______________________________________                                         5 minutes     49.8     57.5                                                    15 minutes 43.3 54.8                                                          30 minutes 28.2 46.3                                                          60 minutes 15.5 41.5                                                           2 hours 8.6 23.4                                                              4 hours 5.2 22.0                                                              6 hours 2.5 20.5                                                             24 hours 0.2 7.2                                                              48 hours 0.1 3.6                                                            ______________________________________                                    

BR96 sFv-PE40 clears from the blood faster than ChiBR96-LysPE40. Theestimated half-life in the blood for the single-chain immunotoxin isapproximately 30 minutes as compared to almost 2 hours for the chimericBR96 immunotoxin conjugate. In this experiment, the measurement of ¹²⁵I-labelled BR96 immunotoxin in the blood determined how much of themolecule was present.

In order to measure the amount of detectable single-chain immunotoxinthat was biologically active, the blood was assayed for BR96 sFv-PE40directed cytotoxic activity at the various times indicated in Table 12.

The results in this Example provide an expression plasmid for theproduction of a single-chain immunotoxin composed of thecarcinoma-reactive antibody of the invention, BR96 and a truncated formof Pseudomonas exotoxin. The chimeric molecule, BR96 sFv-PE40, purifiedfrom E. coli exists in both a monomeric and an aggregated form. Thespecificity of the monomeric BR96 sFv-PE40 for its antigen was confirmedthrough a competition analysis with BR96 IgG. The FACS analysis of fivedifferent cell lines demonstrates the distribution of the BR96 antigen,and the cytotoxic potential of BR96 sFv-PE40 was correlated with therelative number of antigen expressed on the surface of the target cells.BR96 sFv-PE40 is extremely potent against cancer cells displaying theBR96 antigen, with MCF-7 cells being the most sensitive cell lineexamined. BR96 sFv-PE40 was shown to be more potent than the BR96 IgGchemical conjugate against the tumor cell lines tested. To assess thepotential anti-tumor activity of BR96 sFv-PE40, the chimeric toxin wasintravenously administered to mice and found to have a serum half-lifeof 30 minutes, as compared to that of ChiBR96-LysPE40, which was almost2 hours. It may be an advantage that the single-chain immunotoxin iscleared so rapidly from the blood. The immunotoxin molecules are stableand retain biological activity following administration into animals.

EXAMPLE 15 In Vivo Effects of BR96 sFv-PE40

Anti-tumor Activity of BR96 sFv-PE40 Against Human Tumor Xenographs

L2987 and MCF-7 tumor fragments were implanted into female athymic mice(nu/nu) (Harlan Sprague Dawley, Indianapolis, Ind.) at 4-6 weeks of age.They were implanted with L2987 and MCF-7 tumor fragments fromestablished tumor xenografts that were approximately 4 weeks old (800 cumm). Tumor sections were implanted subcutaneously using a trocar ontothe back hind quarter of the mice. Two weeks after implantation theanimals were randomized and their tumors measured.

For the anti-tumor experiments, we only used animals that had tumorsranging from 50-100 cubic mm in size. The animals were intravenouslyinjected via the tail vein with the BR96 sFv-PE40 immunotoxin accordingto the administration schedule indicated in FIGS. 43 and 44. Eachtreatment group consisted of five to ten animals.

Regression of MCF-7 breast carcinoma xenografts was observed with dosesup to 0.75 mg/kg using administration schedules of Q4DX3 (FIG. 43).Using an administration schedule of Q2DX5, the L2987 lung tumors wereobserved to regress upon treatment with BR96 sFv-PE40 (FIG. 44).Complete regression of the tumor xenografts was observed at dosesranging from 0.375 mg/kg to 0.125 mg/kg.

The effect of the tumor xenografts was dose-dependent, as at lower dosesthe tumors were able to grow back after being regressed for a seven dayperiod while at higher doses the complete regression lasted for overtwenty days.

In untreated animals, the tumors grew rapidly and the animals weresacrificed approximately 30 days post implantation. No apparent toxicitywas observed at the doses used in this experiment.

The tumor xenografts used in this study emanated from a small piece of asolid tumor excised from another animal. The tumor tissue wassubcutaneously implanted and allowed to vascularize and grow beforetreatment was initiated. In this manner, the data presented hereindemonstrate a tumor model of tumors found in humans.

EXAMPLE 16 Materials and Methods

Animals

Athymic mice and athymic Rowett rats (Harlan Sprague Dawley) were usedin this study.

The binding of BR96 to normal rat tissues was similar to BR96 binding tonormal human tissues, i.e., BR96 bound to cells in the esophagus,stomach, intestine and acinar cells of pancreas.

In contrast to rats, normal tissues from athymic mice did not bind BR96.

BR96-DOX

The conjugates were prepared by linking the DOX derivativemaleimidocaproyl doxorubicin hydrazone to BR96 or control immunoglobulin(FIG. 45). For more detail, see Examples 20, 22 and 26.

Implanted Carcinoma Cells

L2987 lung carcinoma cells were selected in vitro for the ability togrow as multicellular spheroids. When injected IV into athymic mice orrats, tumors developed at various sites, including lymph nodes, lung,spleen, liver, brain, subcutaneously, and ascites was formed in someanimals.

Athymic rats were transplanted subcutaneously with human lungadenocarcinoma L2987, colon carcinoma RCA, or breast carcinoma MCF7 andpermitted to grow. Therapy (3 treatments 4 days apart) started 14 to 28days after tumor transplantation when tumors were well established, i.e.when the tumors were 50 to 100 mm³ in size.

Administration of BR96 Into The Animals

Mice and rats were administed with BR96-DOX by three injections, eachinjection being about four days apart. Mice which were injectedintraperitoneally (IP) were given 20 mg/kg of BR96-DOX. Mice which wereinjected intravenously were given 10 mg/kg of BR96. Intravenous (IV)injection involved less volume of BR96 because of the constraints ofinjection volume, namely, only 10 mg/kg was administrable by IV.

At the doses tested, there was no difference in the anti-tumor activityof BR96-DOX whether administed IP or IV.

Controls

One set of controls included untreated mice and mice that received (1)DOX (at doses optimized to produce maximal antitumor activity in eachmodel); (2) unconjugated BR96; (3) mixtures of BR96 and DOX; and (4) DOXconjugated to either normal human IgG or the control MAb SN7. Doses ofDOX and MAb are presented as mg/kg/infection.

Another set included rats using the protocol used in control mice.

Results of Treatment of Mice

Treatment with BR96-DOX consistently cured most mice bearing L2987 (FIG.46A) or RCA (FIG. 46B) tumors. Further, mice treated with BR96-DOXexhibited complete and partial tumor regressions against MCF7 tumors(FIG. 46C). Complete tumor regression (CR) refers to a tumor that for aperiod of time is not palpable. Partial tumor regression (PR) means adecrease in tumor volume to ≦50% of the initial tumor volume.Specifically, BR96-DOX cured 78% of the treated mice. In contrast, DOXalone was not active against established RCA tumors either in terms oftumor growth delay or regressions.

The MTD of free DOX (4 mg/kg administered as 3 injections 4 days apart)resulted in a delay in tumor growth and 25% cures. However, BR96-DOXgiven at a matching DOX dose (4 mg/kg DOX, 140 mg/kg BR96) cured 100% ofthe animals.

FIGS. 46A-D are line graphs showing the antigen-specific anti-tumoractivity of BR96-DOX. FIG. 46A shows mice transplanted with L2987 lungtumor xenografts which have grown to about 50 to 100 mm³ at theinitiation of therapy. Treatment with BR96-DOX consistently cured mostmice bearing L2987 (FIG. 46A).

FIG. 46B shows mice transplanted with colon carcinoma RCA which havegrown to tumor xenografts of about 50 to 100 mm³ at the initiation oftherapy. Treatment with BR96-DOX consistently cured most mice bearingcolon carcinoma RCA (FIG. 46B).

FIG. 46C shows the efficacy of BR96-DOX in mice transplanted with MCF7tumors. These mice treated with BR96-DOX consistently exhibited completeand partial tumor regressions against MCF7 tumors (FIG. 46C).

Equivalent doses of non-binding IgG-DOX or SN7-DOX had no effect againstthese tumors. Although optimal doses of DOX delayed the growth of smallL2987 tumors (50 to 100 mm³) and MCF7 tumors; regressions or cures werenot observed.

FIG. 46D shows mice transplanted with L2987 lung tumor xenografts whichgrew to about 250 to 800 mm³ at the initiation of therapy. Such micewhich were treated with BR96-DOX also exhibited 56% cures, 22% completeand 22% partial regressions of lung tumors. In contrast, antitumoractivity was not observed after treatment with an optimal dose of DOX.

FIG. 47 shows that BR96 is efficacious in curing athymic mice havinglarge disseminated tumors.

Mice were inoculated IV with L2987 spheroids. Approximately twelve weekslater mice (14 mice/group) were selected for treatment with BR96 (8mg/kg equivalent DOX administered as 3 injections 4 days apart) or DOXon the basis of visible tumor burden, i.e. therapy was delayed untilmice displayed extensive disseminated disease, ≧0.5 g of visible tumorburden.

The burden of disseminated disease in these animals was so far advancedthat 50% of control animals died during the first 6 days of theexperiment. The median survival time (MST) of the control group was 90days and 100% of the mice were dead by day 102. Surviving mice weresacrificed 200 days after cell inoculation and sections of lung, lymphnodes, spleen, colon, jejunum, kidney, liver, brain, and heart wereexamined by histology.

Mice inoculated with L2987 spheroids and treated had an increased MST(MST of >200 days) relative to that of control mice (MST of 85 days) ormice treated with an optimal dose of DOX (MST of 140 days).

According to immunohistology, the degree of binding of BR96 to cellsfrom these carcinoma lines was similar to that of biopsy material fromhuman carcinomas of the same respective types (21).

Table 13 summarizes the tumor regression rates following treatment withvarious doses of (1) BR96-DOX, (2) DOX, and (3) mixtures of MAb and DOXagainst established L2987 and RCA tumor xenografts in mice.

BR96-DOX administered at equivalent DOX doses of ≧5 mg/kg (3 injections4 days apart) produced long-term cures in 72 to 100% of mice (n=291)bearing L2987 tumors.

In the RCA colon tumor model, which was not sensitive to unconjugatedDOX, BR96-DOX administered at equivalent DOX doses of ≧10 mg/kg (3injections 4 days apart) cured 72 to 100% of mice (n=48).

Mice cured of L2987 or RCA tumors remained alive and tumor free for morethan 1 year with no indication of side effects.

                                      TABLE 13                                    __________________________________________________________________________    Antitumor activity of BR96-DOX against established human tumor xenografts                 Dose         PERCENT                                                                                       (mg/kg/injection) TUMOR REGRESSIO                                            NS* No.                               Treatment                                                                            Schedule                                                                           DOX                                                                              MAb BR96*                                                                           Tumor                                                                             Cures                                                                              Complete                                                                           Partial                                                                            MICE                                  __________________________________________________________________________                         L2987                                                    BR96-DOX                                                                             q4d × 3.sup.+                                                                20.0                                                                             689       100  0    0    8                                         15.0 711 ± 36  83.0 ± 0.8  3.3 ± 0.9  7.0 ± 0.9 29                                                       10.0 513 ± 12  83.0 ±                                                1.1  8.0 ± 0.7  2.0 ± 0.4                                               100                                       8.0 317 ± 3   88.5 ± 0.1  3.7 ± 1.0 0 27                             5.0 246 ± 5   72.3 ± 2.2 17.9 ± 1.5  5.6 ± 0.7 117                                                       2.5 109 ± 3   30.4 ± 3.4                                             33.7 ± 2.4 21.3 ± 2.6 62                                                   1.25 49 ± 1   6.9 ± 0.9                                              11.6 ± 1.2 11.9 ± 2.1 44                                                 BR96-DOX q1d × 1.sup..noteq                                            . 30.0 1078   50.0 50.0 0 10                                                     25.0 930  30.0 30.0 40.0 10                                                   20.0 735  60.0 20.0 10.0 10                                                   15.0 540  11.0 22.0 44.0 9                                                  IgG-DOX q4d × 3 10.0  403                                              ± 5.2  0 0 0 19                        5.0  202 ± 3.2  0 0  3.7 ± 1.0 27                                     DOX q4d × 3 8.0 --  0 0  0.8 ± 0.8 125                               MAb BR96 q4d × 3 -- 400  0 0 0 8                                          -- 200  0 0 0 8                                                               -- 100  0 0 0 8                                                             BR96 + DOX q4d × 3 8.0 400  0 0 0 9                                       8.0 200  0 0 0 9                                                              8.0 100  0 0 0 9                                                                               RCA                                                      BR96-DOX                                                                             q4d × 3                                                                      20.0                                                                             903       100  0    0    10                                        15.0 625  80.0 10.0 10.0 10                                                   10.0 376 ± 5.4  71.7 ± 0.9 0 10.7 ± 0.1 28                           5.0 176  11.0 22.0 11.0 9                                                     2.5  90  0 0  5.5 ± 1.3 18                                               BR96-DOX q7d × 3.sup.@ 20.0 900  100 0 0 10                               15.0 625  100 0 0 10                                                          10.0 420  80 10 0 10                                                          5.0 210  10 0 10 10                                                         IgG-DOX q4d × 3 10.0 405  0 0 0 10                                      DOX q4d × 3 8.0 --  0 0 0 29                                            DOX q7d × 3 10.0 --  0 0 0 10                                         __________________________________________________________________________     *Mean ± SEM                                                                .sup.+ 3 injections administered with a 4 day interval                        .sup.≠ single injection                                                 .sup.@ 3 injections administered with a 7 day interval                   

BR96 administered at equivalent doses was not active against establishedtumors (either in terms of tumor growth delay or regressions) and thetumor growth delay produced by mixtures of BR96 and DOX was equivalentto that of DOX administered alone.

Contrary to our expectations, cells lacking BR96 expression were notdetected after treatment with BR96-DOX. Also, cells obtained from tumorsthat grew back after BR96-DOX induced regression were as sensitive invitro to DOX as the parental cell line.

The IC₅₀ (concentration required to produce 50% inhibition of ³[H]-thymidine incorporation) was 0.4±0.1 μM and 0.3±0.2 μM DOX fortreatment and parental, respectively. These cells were also as sensitiveto BR96-DOX as the parental cell line with IC₅₀ values of 2.7±0.5 μM and2.6±0.8 μM equivalent DOX for parental and treated, respectively.

These data suggest that it may be possible to successfully retreattumors with several rounds of BR96-DOX therapy.

The maximum tolerated dose (MTD) (equivalent DOX dose) of the BR96-DOXconjugate (administered as 3 injections 4 days apart) was 20 mg/kgadministered intraperitoneally (IP). When administered intravenously(IV) the MTD was ≧10 mg/kg. This was the maximum dose that could beadministered IV because of the constraints of injection volume.

At the doses tested, there was no difference in the antitumor activityof BR96-DOX whether administered IP or IV. At doses of BR96-DOX(Table 1) equivalent to ≧mg/kg of DOX (≧250 mg/kg BR96) more than 70% oftreated animals were cured of established L2987 tumors.

In fact, the BR96-DOX (Table 13) equivalent to ≧5 mg/kg of DOX (>250mg/kg BR96) more than 70% of treated animals were cured of establishedL2987 tumors. The BR96-DOX conjugate was active at doses as low as 1mg/kg equivalent DOX. Therefore, the BR96-DOX conjugate was active at adose equivalent to 1/20th of its MTD.

These data demonstrate the broad range of therapeutic doses which wereachieved with BR96-DOX. The MTD of unconjugated DOX (8 mg/kg IV and 4mg/kg IP) was lower than that of the BR96-DOX conjugate. UnconjugatedDOX administered IV at the MTD produced a delay in tumor growth but notumor regressions and if the dose was reduced to 50% of the MTD, DOX hadno effect.

In contrast, activity equivalent to that of an optimal dose of DOX (8mg/kg) was achieved at a dose of 1 mg/kg of BR96-DOX. The BR96-DOXconjugate produced antitumor activity comparable to that of an optimaldose of unconjugated DOX at 1/8th of the equivalent DOX dose. Insummary, the BR96-DOX conjugate was more active, had a much broaderrange of therapeutic doses, and was more potent than unconjugated DOX.

Seven of the 8 surviving mice were free of detectable tumor (70% curesby combined life span and histologic examination).

The BR96-DOX conjugate demonstrated strong antitumor activity in allpreclinical models evaluated. The efficacy and potency of BR96-DOXconjugates is likely due to several factors. The antigen to which BR96binds is abundantly expressed at the tumor cell surface and active drugis released following antigen-specific binding and internalization ofthe conjugate into the acidic environment of lysosomes/endosomes.

Acid-labile immunoconjugates, in which a less stable disulfide linkerwas used, have been investigated previously (7,26). Although theseconjugates were active in an antigen-specific manner, they had poorpotency in vivo (7). The use of a more stable thioether linker, and aMAb with higher avidity and more rapid rates of internalization,improved the activity and potency of BR96-DOX conjugates and alsoincreased the range of therapeutic doses.

We showed that administration of BR96-DOX conjugate at cumulative dosesof at least 15 mg/kg DOX and 700 mg/kg MAb (equivalent to 45 mg/m² DOXand 2100 mg/m² MAb) resulted in greater than 70% cures of establishedlung tumors. This dose of MAb in mice is approximately equivalent to acumulative dose of 3 g of MAb per patient and is only slightly higherthan that required to achieve saturation of human carcinomas in patientsgiven L6, another anticarcinoma MAb (G. Goodman, et al., J. Clin.Oncol., 8, 1083 (1990).

It would be clear to those skilled in the art that the optimal schedulefor administering BR96-DOX will vary based upon the subject, thesubject's height and weight, the severity of the disease.

The demonstration of tumor cures in animals in which BR96 binds tonormal tissues highlights the fact that the appropriate combination ofMAb, drug, and linker chemistry are critical aspects to successfulantibody-directed therapy. The toxic effects of DOX are dose related andit is likely that increasing the intra-tumoral concentration of DOX willproduce a significant increase in antitumor activity (S. K. Carter, J.Natl. Cancer Inst., 55, 1265 (1975); R. C. Young, R. F. Ozols, C. E.Myers, N. Eng. J. Med., 305, 139 (1981)).

BR96-DOX induced complete regressions and cures of xenografted humanlung, breast and colon carcinomas growing subcutaneously in athymic miceand cured 70% of mice bearing extensive metastases of a human lungcarcinoma.

Results of Treatment with Rats

The MTD of free DOX (4 mg/kg administered as 3 injections 4 days apart)resulted in a delay in tumor growth and 25% cures.

The MTD of free DOX (4 mg/kg administered as 3 injections 4 days apart)resulted in a delay in tumor growth and 25% cures. However, BR96-DOXgiven at a matching DOX dose (4 mg/kg DOX, 140 mg/kg BR96) cured 100% ofthe animals, and a dose equivalent to 2 mg/kg DOX (70 mg/kg BR96) cured88% of the rats.

BR96-DOX given at a matching DOX dose (4 mg/kg DOX, 140 mg/kg BR96)cured 100% of the animals, and a dose equivalent to 2 mg/kg GOX (70mg/kg BR96) cured 88% of the rats. Of the rats treated with BR96-DOX,94% (15/16) remained alive and tumor free with no evidence of toxicity150 days after the last dose of BR96-DOX.

It is surprising that BR96-DOX also cured 94% of athymic rats withsubcutaneous human lung carcinoma, even though the rats, like humans, incontrast to mice, express the BR96 target antigen in some normaltissues.

The BR96-DOX conjugate demonstrated antigen-specific activity in vitroand was 8 to 25 fold more potent than non-binding (IgG-DOX or SN7-DOX)conjugates against carcinoma lines that expressed the BR96 antigen.BR96-DOX was much less active against cells that did not bind BR96.

Optimal doses of DOX 8 mg/kg) had no effect on the large disseminatedtumors; the MST was 94 days and 100% of the mice were dead by day 140.In contrast, mice treated with BR96-DOX (8 mg/kg) had a MST of ≧200 daysand 8 of the 10 animals survived for the duration of the experiment.

EXAMPLE 17 Conjugate of SPDP Thiolated Monoclonal Antibody BR64 with theMaleimidocaproylhydrazone of Adriamycin

A solution of the BR64 antibody (25 mL, 10.37 mg/mL; determined by UV at280 nm, 1.4 absorbance units equal 1 mg protein) was treated with SPDPsolution in absolute ethanol (1.3 mL of 10 mmol solution). The solutionwas incubated for 1 hour at 31°-32° C., then chilled in ice and treatedwith a solution of DTT in phosphate buffered saline ("PBS") (1.3 mL of a50 mmol solution). The solution was kept in ice for 1 hour thentransferred to a dialysis tube and dialyzed three times against PBS (2 Lper dialysis) for a period of at least 8 hours. After the dialysis, theconcentration of protein was measured, as above, followed by adetermination of molar concentration of free sulfhydryl groups by theEllman method.

The thiolated protein (3 mL) was treated with an equivalent thiol molaramount of maleimidocaproylhydrazone of adriamycin, prepared as inPreparation 2, dissolved in dimethylformamide (DMF) (5 mg/mL, 0.131 mL)and the mixture was incubated at 4° C. for 24 hours. The solution wasdialyzed three times against PBS (1000 mL) for a period of at least 8hours. The solution was centrifuged and the supernatant was shaken for afew hours with Bio-beads™ SM-2 (non-polar neutral macroporouspolystyrene polymer beads, Bio-Rad Laboratories, Richmond, Calif. 94804)and finally filtered through a Millex-GV (Millipore Corporation,Bedford, Mass. 01730) 0.22 μm filter unit. The overall average number ofmolecules of adriamycin per molecule of antibody ("MR") was determinedby measuring the amount of adriamycin from the absorption at 495 nm(ε=8030 cm⁻¹ M⁻¹) and the amount of protein from the absorption at 280nm, after correcting for the absorption of adriamycin at 280 nmaccording to the formula: ##EQU2##

The MR found for the product was 5.38; free adriamycin 0.14%; proteinyield 60%.

EXAMPLE 18 Conjugate of SPDP Thiolated BR64 with theMaleimidocaproylhydrazone of Adriamycin

A solution of the BR64 antibody (405 mL, 11.29 mg/mL) was stirred andtreated with SPDP solution in absolute ethanol (22.3 mL of 10 mmolsolution). The solution was incubated for 1 hour at 31°-32° C. whilebeing gently shaken, then cooled in ice to 4° C., stirred and treatedwith a solution of DTT in PBS (22.3 mL of a 50 mmol solution). Thesolution was kept in ice for 1 hour then divided into 2 equal parts,each transferred to a dialysis tube and dialyzed six times against PBS(6 L per dialysis) for a period of at least 8 hours. After that thecontents of the tubes were combined (400 mL) and the concentration ofprotein and free thiol groups was determined (molar ratio of --SH groupsto protein is 8.5).

The solution of thiolated protein was stirred and treated with anequivalent thiol molar amount of maleimidocaproyl hydrazone ofadriamycin dissolved in DMF (5 mg/mL, 35.7 mL) and the mixture wasincubated at 4° C. for 24 hours. The solution was divided into 2 equalparts, transferred to dialysis tubes and dialyzed five times against PBS(6 L per dialysis) for a period of at least 8 hours. The contents of thedialysis tubes were combined, filtered through a 0.22μ cellulose acetatefilter, and the filtrate was shaken for 24 hours with Bio-beads™ SM-2(Bio-Rad Laboratories, Richmond, Calif. 94804). The solution wasfiltered through a 0.22μ cellulose acetate filter. The concentration ofprotein and adriamycin was determined (6.26 mg/mL and 162.4 μg/mL,respectively) yielding a molar ratio (MR) of 7.18. The protein yield was77%. Unconjugated adriamycin present was 0.07%.

EXAMPLE 19 Conjugate of SPDP Thiolated SN7 with theMaleimidocaproylhydrazone of Adriamycin

In a manner analogous to that described in Examples 17 and 18 monoclonalantibody SN7, an antibody which does not bind to the antigen recognizedby BR64, was thiolated with SPDP and reacted with the maleimidocaproylhydrazone of adriamycin to yield a conjugate with a molar ratio (MR) of4. Protein yield was 51%. Unconjugated adriamycin present was 0.36%.

EXAMPLE 20 Conjugate of SPDP Thiolated ChiBR96 with theMaleimidocaproylydrazone of Adriamycin

A solution of chimeric BR96 antibody, ChiBR96, (27.5 mL, 12.53 mg/mL)was treated with a 10 mM solution of SPDP in absolute ethanol (1.7 mL).The solution was incubated at 31° C. for 35 minutes, chilled in ice andtreated with a 0.50 mM solution of DTT in PBS (1.7 mL) for 15 min at 4°C. The solution was transferred to a dialysis tube and dialyzed fourtimes in PBS-0.1 M histidine buffer (4.5 L per dialysis) for a period ofat least 8 hours. The amount of protein and molar concentration of thiolgroups was determined (9.29 mg/mL and 2.06×10⁻⁴ M, respectively). Thesolution (17 mL) was treated with an equivalent molar amount of themaleimidocaproylhydrazone of adriamycin in DMF (5 mg/mL, 0.59 mL) andthe reaction mixture incubated at 4° C. for 24 hours. The reactionmixture was dialyzed three times, in the same buffer (4.5 L perdialysis), for at least 8 hours. The dialyzed solution was centrifugedand the supernatant shaken gently with Biobeads™ SM-2 (Bio-RadLaboratories, Richmond, Calif. 94804) for a few hours at 4° C. Thesolution was centrifuged and the concentration of protein and adriamycinin the supernatant (19 mL) was determined (6.5 mg/mL and 67.86 μg/mL,respectively). The molar ratio of drug to protein is 2.9. Protein yieldis 72% ; unconjugated adriamycin present is 1.2%.

EXAMPLE 21 Conjugate of Modified Bombesin with theMaleimidocaproylhydrazone of Adriamycin

Bombesin does not contain a free reactive sulfhydryl group which can beused to link the drug through the Michael Addition Receptor-containinglinker. Thus, there was prepared a modified bombesin which contains anadditional cysteine residue at the amino terminus of native bombesin. Inaddition, residue-3 of the native bombesin has been changed to a lysineresidue. The modified bombesin, therefore, is designated "Cys⁰ -lys³-bombesin".

Cys⁰ -lys³ -bombesin (11.3 mg) was dissolved in 1.1 mL of deionizedwater and adjusted to pH 7-7.5 with 10 μl 1.5 M Tris-HCl, pH 8.8 andthen reacted with 0.45 mL maleimidocaproyl adriamycin hydrazone (15mg/mL in deionized water) at ambient temperature for several hours. Thereaction mixture was dialyzed against water overnight in dialysis tubing(molecular weight cutoff: 1000). The precipitate was removed bycentrifugation (12,000× g) and the supernatant was saved. Adriamycin("ADM") content of the bombesin-adriamycin conjugate was measured bydiluting 1:50 in acetate buffer, pH 6.0. The adriamycin ("ADM") contentwas calculated using the formula:

    [O.D..sub.495 /8030]×50=ADM (M)

For this preparation O.D.₄₉₅ =0.116 thus the adriamycin content was7.2×10⁻⁴ M.

The product was chromatographed by HPLC using a C₁₈ (BeckmanInstruments, Ultrasphere 5μ, 4.6 mm×25 cm) column. Buffer A: 10 mM NH₄OAc pH, 4.5; Buffer B: 90% acetronitrile/10% Buffer A. The column wasequilibrated with 90% Buffer A/10% Buffer B and the chromatographyconditions were: 90% buffer A/10% buffer B to 60% Buffer A/60% buffer Bfor 2 minutes, gradient to 50% buffer A/50% buffer B for 15 minutes. Theproduct had a retention time of 9.3 minutes under these conditions.

EXAMPLE 22 A Conjugate of Iminothiolane Thiolated Chimeric BR96 andMaleimidocaproyl Hydrazone of Adriamycin

Chimeric BR96 (15 mL, 9.05 mg/mL) was dialysed two times against 4liters of 0.1 M sodium carbonate/bicarbonate buffer, pH 9.1. Theantibody solution then was heated with iminothiolane (0.75 mL, 20 mM) at32° C. for 45 minutes. The solution was then dialysed against 4 litersof sodium carbonate/bicarbonate buffer, pH 9.1 followed by dialysisagainst 4 liters of 0.0095 M-PBS-0.1 M L-histidine, pH 7.4. Thissolution had a molar ratio of --SH/protein of 1.35. The protein then wasre-thiolated as described above to yield a solution with a molar ratioof --SH/protein of 5.0.

The maleimidocaproyl hydrazone of adriamycin (3.2 mg in 0.640 mL DMF)was added with stirring at 4° C. to the thiolated protein solution. Theconjugate was incubated at 4° C. for 16 hrs then it was dialysed against4 liters of 0.0095 M PBS-0.1 M L-histidine, pH 7.4. The dialysedconjugate was filtered through a 0.22μ cellulose acetate membrane into asterile tube to which a small quantity (>5% (v/v)) of BioBeads™ SM-2(Bio-Read Laboratories, Richmond, Calif. 94804) were added. After 24 hrsof gentle agitation, the beads were filtered off and the conjugate wasfrozen in liquid nitrogen and stored at -80° C. The resulting conjugatehad a molar ratio of 3.4 adriamycin molecules to 1 molecule of proteinand was obtained in 24% yield from chimeric BR96.

EXAMPLE 23 Conjugate of Maleimidocaproyl Hydrazone of Adriamycin withDTT reduced Human IgG ("Relaxed Human IgG")

Human IgG (obtained from Rockland, Gilbertsville, Pa.) was diluted with0.0095 M PBS to a protein concentration of 10.98 mg/mL. This solution(350 mL) was heated to 37° C. in a water bath under a nitrogenatmosphere. Dithiothreitol (16.8 mL, 10 mM) in PBS was added and thesolution was stirred for 3 hrs at 37° C. The solution was dividedequally between two Amicon (Amicon Division of W. R. Grace and Co.,Beverly, Mass. 01915) Model 8400 Stirred Ultrafiltration Cells, eachfitted with an Amicon YM 30 Ultrafilter membrane (MW cutoff 30,000, 76mm diam.) and connected via an Amicon Model CDS10 concentration/dialysisselector to an Amicon Model RC800 mini-reservoir. Each reservoircontained 700 mL of 0.0095 M PBS-0.1 M L-histidine. The proteinsolutions were dialyzed until concentration of free thiol in thefiltrate was 41 μM. The molar ratio of --SH/protein in the retentate wasdetermined to be 8.13.

The retentate was transferred from the cells to a sterile containermaintained under a nitrogen atmosphere and a solution ofmaleimidocaproyl hydrazone of adriamycin (36.7 mL, 5 mg/mL in water) wasadded with stirring. The conjugate was incubated at 4° C. for 48 hrsafter which it was filtered through a 0.22μ cellulose acetate membrane.A Bio-Rad Econocolumn™ (2.5 cm×50 cm, Bio-Rad Laboratories, Richmond,Calif. 94804) was packed with a slurry of 100 g of BioBeads™ SM-2(Bio-Rad Laboratories, Richmond, Calif. 94804) in 0.00095 M-0.1 ML-histidine buffer. The beads had been prepared by washing in methanol,followed by water and then several volumes of buffer. The filteredconjugate was percolated through this column at 2 mL/min. Afterchromatography the conjugate was filtered through a 0.22μ celluloseacetate membrane and frozen in liquid nitrogen and stored at -80° C. Theconjugate obtained had an average molar ratio of 7-45 molecules ofadriamycin per molecule of protein and was obtained in 99% yield fromhuman IgG.

EXAMPLE 24 Conjugate of Relaxed BR64 with Maleimidocaproyl Hydrazone ofAdriamycin

A solution of BR64 (435 mL; 11.31 mg/mL, 7.07×10⁻⁵ M) was treated withDTT (947 mg) and heated at 42°-43° C. with gentle stirring for 2 hrs.The solution was cooled in ice, transferred into 2 dialysis tubes andeach tube was dialyzed 5 times against PBS (14 L per dialysis) for 8 hrsat 4° C. The contents of the tubes were combined (400 mL) and theprotein and --SH content determined (10.54 mg/mL, 6.58×10⁻⁵ M; 5.14×10⁻⁴M, respectively). The molar ratio of --SH to protein was 7.8.

A solution of maleimidocaproyl hydrazone of adriamycin in DMF (5 mg/mL,32.6 mL) was added to the antibody solution with gentle stirring andthen incubated at 4° C. for 24 hrs. The solution was filtered through a0.22μ cellulose acetate filter and then transferred to two dialysistubes and dialyzed as described above. After dialysis, the contents ofthe tubes were combined, filtered and shaken with BioBeads™ SM-2(Bio-Rad Laboratories, Richmond, Calif. 94804) for 24 hrs at 4° C. Thebeads were filtered off using a cellulose acetate filter to yield theconjugate solution. The concentration of protein and adriamycin weredetermined (8.66 mg/mL, 5.42×10⁻⁵ M; 168 μg/mL, 2.89×10⁻⁴ M,respectively). The protein yield is 97%. The molar ratio of adriamycinto protein is 5.33; and, unconjugated adriamycin is 0.5%.

EXAMPLE 25 General Procedure for Conjugating theMaleimidocaproylhydrazone of Adriamycin to a Relaxed Antibody

1. A solution (300 mL) of antibody (3 g, 10 mg/mL) in PBS buffer iscontinuously blanketed with nitrogen, immersed in a 37° C. water bathand stirred gently with a magnetic stirrer. The solution is treated with7 molar equivalents of DTT for 3 hrs. The --SH group molar ratio ("MR")to protein is determined initially and hourly and, for a maximallyconjugate product, should remain constant at about 14.

2. The solution is transferred as quickly as possible to an Amicondiafiltration cell (Amicon, Division of W. R. Grace & Co., Beverly,Mass. 01915) (note 1) maintained at about 4° C. to about 7° C. Thesystem is pressurized with argon or nitrogen and diafiltration isstarted using PBS buffer containing 0.1 M histidine which had beenprecooled to about 4° C. to about 7° C.). The initial temperature of theeffluent, immediately after starting the diafiltration, is 16-18° C. anddrops to 8°-9° C. within about 90 minutes. The effluent is monitored fora MR of --SH to protein and, when this value is <1, the diafiltration iscomplete (note 2).

3. The solution is transferred back to a round bottom flask equippedwith a magnetic stirrer and kept in ice. The solution continuously isblanketed by nitrogen. The volume of the solution is noted. Aliquots of0.1 mL are taken out and diluted with PBS buffer to 1.0 mL to determinethe amount of protein in mg/mL (and also the molar equivalent of proteinand the molarity of the --SH groups (and hence the MR of the --SH toprotein). A solution of maleimidocaproylhydrazone of adriamycin indistilled water (5 mg/mL, 6.3×10⁻³ M) is prepared (note 3, 4). Theamount (in mL) of this solution needed for conjugation is determined bythe formula: ##EQU3## (note 5) and this amount is added slowly to theprotein solution which is stirred gently. The solution is kept a 4° C.for 30 min.

4. A column of Bio-Beads™ SM-2, mesh 20-50 (Bio-Rad Laboratories,Richmond, Calif. 94804) is prepared (note 6) at 4° C. The red proteinsolution is filtered through a 0.22μ cellulose acetate filter, thenpassed through the column at a rate of 2.5 mL/min and the red effluentcollected. Finally PBS-0.1 M histidine buffer is poured on top of thecolumn and the effluent collected until it is colorless. The volume ofthe collected red solution is noted. An aliquot of 0.1 mL is diluted to1 mL with PBS buffer and the amount of protein and adriamycin ismeasured. The amount of conjugated adriamycin is determined byabsorbance at 495 nm (ε=8030 cm⁻¹ M⁻¹) and expressed in micromoles andmicrograms per mL. The amount of protein, expressed in mg per mL andmicromoles, is determined as above by reading the absorbance at 280 nmwith a correction for the absorbance of adriamycin at the samewavelength according to the general formula ##EQU4## where A is theobserved absorbance at the noted wavelength. The MR of adriamycin toprotein then is calculated.

5. An aliquot of 5 mL of conjugate is passed over an Econo-Pac™ 10 SM-2column (a prepacked Bio-Beads™ SM-2 column (Bio-Rad Laboratories,Richmond, Calif. 94804), volume 10 mL, that has been washed andequilibrated with PBS-0.1 M histidine buffer) in the manner describedabove. The amount of protein and conjugated adriamycin is determined andthe MR determined. This value should be the same as that of the bulk ofthe solution (note 7).

6. The conjugate is frozen in liquid nitrogen and stored at -80° C.Aliquots can be taken for determining cytotoxicity, binding and presenceof free adriamycin (note 8).

1. On a scale of 3 g/300 mL, two Amicon cells of 350 mL each are used,dividing the solution into two portions of 150 mL per cell.

2. On the reaction scale provided, the diafiltration usually takes 2-4hrs. The duration will depend on factors such as the age of themembrane, rate of stirring of solution and pressure in cell.

3. The hydrazone is not very soluble in PBS and a precipitate is formedin a short while.

4. Brief applications of a sonicator will facilitate dissolution indistilled water. The resulting solution is stable.

5. This amount provides for a 5% excess of the hydrazone. The processdescribed generally takes about 15-20 minutes.

6. The Bio-Beads™ are prepared for chromatography by swelling them inmethanol for at least one hr., preferably overnight, washing them withdistilled water and finally equilibrating them with PBS-0.1 M histidinebuffer. For 3 g of protein 100 g of beads are used to form a column of2.5 cm×30 cm.

7. Because of the inherent error of the spectroscopic methods used, avariation of 1 MR unit is accepted to be a satisfactory result.Generally, however, the MR varies less than 0.5 MR units.

8. The values of free adriamycin in the conjugate are generally muchless than 1%.

EXAMPLE 26 Conjugate of Relaxed Chimeric BR96 with MaleimidocaproylHydrazone of Adriamycin

Chimeric BR96, prepared in the manner previously described, was dilutedwith 0.0095 M PBS to a protein concentration of 10.49 mg/mL. Thissolution (500 mL) was heated to 37° C., under a nitrogen atmosphere, ina water bath. Dithiothreitol (26.2 mL, 10 mM) in PBS was added and thesolution was stirred for 3 hrs at 37° C. The solution was dividedequally between two Amicon Model 8400 stirred ultrafiltration cells eachfitted with a YM 30 ultrafilter (MW cutoff 30,000, 76 mm diam.) andconnected via a Model CDS10 concentration/dialysis selector to a ModelRC800 mini-reservoir (Amicon, Division of W. R. Grace and Co., BeverlyMass. 01915-9843). Each reservoir contained 800 mL of 0.0095 M PBS-0.1 ML-histidine. The protein solutions were dialyzed until the concentrationof free thiol in the filtrate was 63 μM. The molar ratio of --SH/proteinin the retentate was determined to be 8.16. The retentate wastransferred from the cells to a sterile container under nitrogen and asolution of maleimidocaproyl hydrazone of adriamycin (42.6 mL, 5 mg/mLin water) was added with stirring. The conjugate was incubated at 4° C.for 48 hrs after which it was filtered through a 0.22μ cellulose acetatemembrane. A 2.5 cm×50 cm Bio-Rad Econocolumn was packed with a slurry of100 g of BioBeads™ SM-2 (Bio-Rad Laboratories, Richmond Calif. 94804) in0.0095 M-0.1 M L-histidine buffer. The beads had been prepared bywashing in methanol, followed by water then several volumes of buffer.The filtered conjugate was percolated through this column at 2 mL/min.After chromatography the conjugate was filtered through a 0.22μcellulose acetate membrane, frozen in liquid nitrogen and stored at -80°C. The conjugate obtained had a molar ratio of 6.77 Adriamycin toprotein and was obtained in 95% yield from chimeric BR96.

EXAMPLE 27 Conjugate of Relaxed Murine Antibody L6 with MaleimidocaproylHydrazone of Adriamycin

Murine antibody L6, prepared as defined earlier, was diluted with 0.0095M PBS to a protein concentration of 11.87 mg/mL. This solution (350 mL)was heated to 37° C., under a nitrogen atmosphere, in a water bath.Dithiothreitol (18.2 mL, 10 mM) in PBS was added and the solution wasstirred for 3 hrs at 37° C. The solution was divided equally between twoAmicon Model 8400 stirred ultrafiltration cells each fitted with a YM 30ultrafilter (MW cutoff 30,000, 76 mm diam.) and connected via a ModelCDS10 concentration/dialysis selector to a Model RC800 mini-reservoir(Amicon, Division of W. R. Grace and Co., Beverly Mass. 01915-9843).Each reservoir contained 800 mL of 0.0095 M PBS-0.1 M L-histidine. Theprotein solutions were dialyzed until concentration of free thiol in thefiltrate was 14 μM. The molar ratio of --SH/protein in the retentate wasdetermined to be 9.8. The retentate was transferred from the cells to asterile container under nitrogen and a solution of maleimidocaproylhydrazone of adriamycin (40.4 mL, 5 mg/mL in water) was added withstirring. The conjugate was incubated at 4° C. for 48 hrs after which itwas filtered through a 0.22μ cellulose acetate membrane. A 2.5 cm×50 cmBio-Rad Econocolumn was packed with a slurry of 100 g of BioBeads™ SM-2(Bio-Rad Laboratories, Richmond, Calif. 94804) in 0.00095 M-0.1 ML-histidine buffer. The beads had been prepared by washing in methanol,followed by water then several volumes of buffer. The filtered conjugatewas percolated through this column at 2 mL/min. After chromatography theconjugate was filtered through a 0.22μ cellulose acetate membrane,frozen in liquid nitrogen and stored at -80° C. The conjugate obtainedhad a molar ratio of 7.39 Adriamycin to protein and was obtained in 100%yield from murine L6.

Biological Activity

Representative conjugates of the present invention were tested in bothin vitro and in vivo systems to determine biological activity. In thesetests, the potency of conjugates of cytotoxic drugs was determined bymeasuring the cytotoxicity of the conjugates against cells of humancancer origin. The following describes representative tests used and theresults obtained. Throughout the data presented, the conjugates arereferred to using the form ligand-drug-molar ratio of ligand to drug.Thus, for example, "BR64-ADM-5.33", refers to a conjugate betweenantibody BR64 and adriamycin and the mole ratio of drug to antibody is5.33. One skilled in the art will recognize that any tumor lineexpressing the desired antigen could be used in substitution of thespecific tumor lines used in the following analyses.

TEST I In vitro Activity of BR64-Adriamycin Conjugates

The immunoconjugates of Examples 18 and 19 were tested in vitro againsta human lung carcinoma line, L2987 [obtained from I. Hellstrom.Bristol-Myers Squibb Seattle; See also I. Hellstrom, et al., CancerResearch 50: 2183 (1990)], which expresses the antigens recognized bymonoclonal antibodies BR64, L6 and BR96. Monolayer cultures of L2987cells were harvested using trypsin-EDTA (GIBCO, Grand Island, N.Y.), andthe cells counted and resuspended to 1×10⁵ /mL in RPMI-1640 containing10% heat inactivated fetal calf serum ("RPMI-10% FCS"). Cells (0.1mL/well) were added to each well of 96-well flat bottom microtiterplates and incubated overnight at 37° C. in a humidified atmosphere of5% CO₂. Media was removed from the plates and serial dilutions ofadriamycin or the antibody conjugates of adriamycin were added to thewells. All dilutions were performed in quadruplicate. Following a 2 hrdrug or conjugate exposure, the plates were centrifuged (100× g, 5 min),the drug or conjugate removed, and the plates washed three times withRPMI-10%FCS. The cells were cultured in RPMI-10% FCS for an additional48 hours. At this time the cells were pulsed for 2 hour with 1.0μCi/well of ³ H-thymidine (New England Nuclear, Boston, Mass.). Theplates were harvested and the counts per minute ("CPM") determined.Inhibition of proliferation was determined by comparing the mean CPM fortreated samples with that of the untreated controls. The data presentedin FIG. 51 demonstrates the cytotoxicity against L2987 lung cells ofbinding immunoconjugate (MR of adriamycin to BR64 equal to 7.18,designated "BR64-THADMHZN-7.18") compared to a non-bindingimmunoconjugate of SN7 and adriamycin (MR of adriamycin to SN7 equal to4, designated "SN7-THADMHZN-4"). The BR64 conjugates prepared by themethod described in Example 18 are active and demonstrateantigen-specific cytotoxicity in this in vitro screen.

TEST II In Vivo Activity of Br64-Adriamycin Conjugates

The immunoconjugates of Examples 18 and 19 were evaluated in vivo forantigen-specific antitumor activity. Congenitally athymic female mice ofBALB/c background (BALB/c nu/nu; Harlan Spraque-Dawley, Indianapolis,Ind.) were used in these studies. Mice were housed in Thoren cagingunits on sterile bedding with controlled temperature and humidity.Animals received sterile food and water ad libitum. The L2987 human lungtumor line, described above, was used in these studies. This line hasbeen shown to maintain expression of the BR64, BR96 and L6 antigensfollowing repeated passage in vivo. The tumor lines were maintained byserial passage in athymic mice as described previously (P. A. Trail, etal., in vivo 3: 319-24 (1989)). Tumors were measured, using calipers, in2 perpendicular directions at weekly or biweekly intervals.

Tumor volume was calculated according to the equation: ##EQU5## in whichV=volume (mm³)

L=measurement of longest axis (mm)

W=measurement (mm) of axis perpendiculars to L.

Data are presented as the median tumor size for treated and controlgroups. Each treatment or control group contained 8-10 animals. Therapywas initiated when tumors had reached a medium size of 50-100 mm³.Therapy was administered by the ip or iv route on various schedule asdenoted. Adriamycin was diluted in normal saline and native antibody andadriamycin conjugates were diluted in phosphate buffered saline ("PBS")for administration. All dosages were administered on a weight basis(mg/kg) and were calculated for each animal. In these studies theantitumor activity of binding BR64 immunoconjugates was compared to thatof optimized dosages of adriamycin, mixtures of native BR64 andadriamycin, and non-binding conjugates. Unconjugated adriamycin wasadministered according to the route, dosage, and schedule demonstratedto be optimal for the L2987 human xenograft model. The unconjugatedadriamycin, therefore, was administered at a dose of 8 mg/kg by the ivroute every fourth day for a total of 3 injections (denoted "8 mg/kg,q4dx3, iv"). The binding (BR64) and non-binding (SN7) immunoconjugateswere administered at several doses by the ip route every fourth day fora total of 3 injections (denoted "q4dx3, ip"). As shown in FIG. 52significant antitumor activity was observed following the administrationof tolerated doses (10 and 15 mg/kg/injection) of the BR64-adriamycinconjugate. The antitumor activity observed following therapy with theBR64 conjugate was significantly better than that observed for therapywith optimized adriamycin and matching doses of a non-binding (SN7)conjugate.

In this experiment, complete tumor regressions were observed in 66% ofthe animals following treatment with 15 mg/kg/injection of the BR64conjugate and 50% complete tumor regressions were observed followingtreatment with 10 mg/kg/injection of the BR64 conjugate. Partial orcomplete regressions of established L2987 tumors have not been observedfollowing therapy with optimized adriamycin, mixtures of native BR64 andadriamycin, or equivalent doses of non-binding conjugates.

To demonstrate that the observed activity required the covalent couplingof the antibody to adriamycin, several control experiments usingmixtures of native BR64 and adriamycin were performed. Representativedata for several types of combined therapy are shown in FIGS. 5a-c. Theantitumor activity observed for various modes of combined therapy withMab and adriamycin was not significantly different from that observedfor therapy with optimized adriamycin alone. Taken together these dataindicate that the covalent coupling of BR64 to adriamycin is required toobserve the antitumor activity described in FIG. 21.

TEST III In Vivo Activity of Bombesin Conjugates

The conjugate of Example 21 was evaluated in vivo for antitumoractivity. BALB/c athymic nude mice were implanted with H345 human smallcell lung carcinoma tumor pieces (obtained from Dr. D. Chen, Universityof Colorado Medical School, CO), subcutaneously, using trocars. Tumorswere allowed to grow to 50-100 mm³ before initiation of treatment. Micewere treated i.v. on 23, 26, 28, and 30 days post-implant withadriamycin alone (1.6 mg/kg), or the conjugates bombesin-adrimycin("BN-ADM(TH)", in an amount equivalent to 1.6 mg/kg adriamycin) orP77-adriamycin conjugate ("P77-ADM(TH)", in an amount equivalent to 1.6mg/kg of adriamycin). P77 is a 12 amino acid peptide with an internalcysteine residue (sequence=KKLTCVQTRLKI) that does not bind to H345cells and was conjugated to the maleimidocaproylhydrazone of adriamycinaccording to the procedure outlined in Example 21. Thus, the conjugaterepresents a non-binding conjugate with respect to H345 cells. Tumorswere measured with calipers and tumor volume was calculated usingformula: ##EQU6## in which V, L, and W are as defined in Test II.

The median tumor volumes were determined and the observed results areshown in FIG. 54.

TEST IV In Vitro Cytotoxicity Data for Relaxed ChiBR96 AntibodyConjugates

Immunoconjugates of adriamycin an ChiBR96 antibody are prepared usingthe general method for preparing relaxed antibodies as described inExample 25. The conjugates were tested, using the protocol below, for invitro cytotoxicity and their cytotoxicity was compared to that of freeadriamycin, and SPDP-thiolated immunoconjugates prepared by the methoddescribed in Example 18. The results of these tests are provided in FIG.55.

Monolayer cultures of L2987 human lung cells were maintained inRPMI-1640 media containing 10% heat inactivated fetal calf serum(RPMI-10%FCS). The cells were harvested using trypsin-EDTA (GIBCO, GrandIsland, N.Y.), and the cells counted and resuspended to 1×10⁵ /ml inRPMI-10%FCS. Cells (0.1 ml/well) were added to each well of 96 wellmicrotiter plates and incubated overnight at 37° C. in a humidifiedatmosphere of 5% CO₂. Media was removed from the plates and serialdilutions of adriamycin or antibody/ADM conjugates added to the wells.All dilutions were performed in quadruplicate. Following a 2 hr drug orconjugate exposure, the plates were centrifuged (200× g, 5 min), thedrug or conjugate removed, and the plates washed 3× with RPMI-10%FCS.The cells were cultured in RPMI-10%FCS for an additional 48 hr. At thistime the cells were pulsed for 2 hr with 1.0 μCi/well of ³ H-thymidine(New England Nuclear, Boston, Mass.). The plates were harvested and thecounts per minute ("CPM") were determined. Inhibition of proliferationwas determined by comparing the mean CPM for treated samples with thatof the untreated control. IC₅₀ values are reported as μM of equivalentadriamycin.

TEST V In Vivo Antitumor Activity of BR64 and Murine L6 Conjugates

The in vivo antitumor activity of immunoconjugates of adriamycin andrelaxed BR64 or relaxed L6 was evaluated. The observed data are providedin FIG. 56.

Congenitally athymic female mice of BALB/c background (BALB/c nu/nu;Harlan Sprague-Dawley, Indianapolis, Ind.) were used. Mice were housedin Thoren caging units on sterile bedding with controlled temperatureand humidity. Animals received sterile food and water ad libitum.

The L2987 human tumor line was established as tumor xenograft models inathymic mice. The tumor line was maintained by serial passage in vivo.Tumors were measured in 2 perpendicular directions at weekly or biweeklyintervals using calipers. Tumor volume was calculated according to theequation: ##EQU7## in which: V=volume (mm³)

L=measurement of longest axis (mm)

W=measurement of axis perpendicular to L

In general, there were 8-10 mice per control or treatment group. Dataare presented as median tumor size for control or treated groups.Antitumor activity is expressed in terms of gross log cell kill ("LCK")where: ##EQU8## T-C is defined as the median time (days) for treatedtumors to reach target size minus the median time for control tumors toreach target size and TVDT is the time (days) for control tumors todouble in volume (250-500 mm³). Partial tumor regression ("PR") refersto a decrease in tumor volume to ≦50% of the initial tumor volume;complete tumor regression ("CR") refers to a tumor which for a period oftime is not palpable; and cure is defined as an established tumor whichis not palpable for a period of time ≧10 TVDTs.

For animals bearing the L2987 human lung tumor, therapy was typicallyinitiated when the median tumor size was 75 mm³ (12-14 days after tumorimplant). The average TVDT was 4.8-0.9 days and antitumor activity wasassessed at a tumor size of 500 mm³. In several experiments (describedbelow in Test VI) therapy was initiated when L2987 tumors were 225 mm³in size.

Materials under investigation were administered by the ip or iv route.Adriamycin was diluted in normal saline; antibody andantibody/adriamycin conjugates were diluted in phosphate bufferedsaline. Compounds were administered on a mg/kg basis calculated for eachanimal, and doses are presented as mg/kg of equivalentadriamycin/injection. Immunoconjugates were administered on a q4dx3schedule. The maximum tolerated dose ("MTD") for a treatment regimen isdefined as the highest dose on a given schedule which resulted in ≦20%lethality.

In the data shown in FIG. 56, injection of optimized doses of adriamycinproduced antitumor activity equivalent to 1.1 LCK and tumor regressionswere not observed. The BR64-ADM conjugate produced antitumor activityequivalent to >10 LCK at all doses tested and 89%, 78%, and 100% cureswere observed at doses of 5 mg/kg, 8 mg/kg, and 10 mg/kg of BR64-ADM,respectively. At doses of 8 mg/kg or 10 mg/kg the L6-ADM conjugateproduced antitumor activity (1.8 and 3.5 LCK, respectively) which wassignificantly better than that of optimized adriamycin but less thanthat of equivalent doses of internalizing BR64-ADM conjugates. Thus, thedata show that the antitumor activity of binding non-internalizingL6-ADM conjugates is superior to that of unconjugated adriamycin.Treatment with L6-adriamycin conjugate results in lower antitumoractivity than is observed with matching doses of the internalizingBR64-adriamycin conjugate.

TEST VI In Vivo Antitumor Activity of ChiBR96-ADM Conjugates

The antitumor activity of ChiBR96-ADM conjugates was evaluated againstestablished human lung ("L2987"), breast ("MCF7", obtainable from theATCC under the accession number ATCC HTB 22; see also I. Hellstrom, etal., Cancer Research 50: 2183 (1990)), and colon ("RCA" from M.Brattain, Baylor University; See also I. Hellstrom, et al., CancerResearch 50: 2183 (1990)) tumors.

Animals were maintained and tumor xenograft models were established forthe MCF7 and RCA and the L2987 human tumor lines as described for theL2987 in Test V. Materials were administered as described in Test V.

For animals bearing the L2987 human lung tumor, therapy typically wasinitiated when the median tumor size was 75 mm³ (12-14 days after tumorimplant). The average TVDT was 3.8±0.9 days and antitumor activity wasassessed at a tumor size of 500 mm³. In several experiments therapy wasinitiated with L2987 tumors were 225 mm³ in size.

The MCF7 tumor is an estrogen-dependent human breast tumor line. Athymicmice were implanted with 0.65 mg (65 day release rate) estradiol pellets(Innovative Research of America, Toledo, Ohio) on the day of tumorimplant. Therapy was initiated when the median tumor size was 100 mm³(typically 13 days after tumor implant). The MCF7 tumor had an averageTVDT of 6.4±2.0 days and antitumor activity was assessed at 500 mm³.

For animals bearing the RCA colon tumor, therapy was initiated 15 daysafter tumor implant when the median tumor size was 75 mm³. The averageTVDT for RCA tumor xenografts was 9.5±1.5 days and antitumor activitywas assessed at 400 mm³. Data for the antitumor activity of optimizedadriamycin in the L2987, MCF7, and RCA xenograft models is summarized inthe following Tables and referenced Figures.

The antitumor activity of the ChiBR96-ADM conjugates was compared tothat of optimized adriamycin and equivalent doses of non-binding (IgG)immunoconjugates. In each model, complete tumor regressions and/or curesof established tumors were observed following the administration oftolerated doses of ChiBR96-ADM conjugate.

Representative data demonstrating the antigen-specific antitumoractivity of ChiBR96-ADM conjugates is presented in FIGS. 57 and 58. Asshown in FIG. 57, the ip administration of ChiBR96-ADM conjugate(MR=4.19) at a dose of 10 mg/kg equivalent of adriamycin producedantitumor activity equivalent to >10 LCK. At this dose of ChiBR96-ADMconjugate, 78% of the mice were cured of the tumor and an additional 11%of mice demonstrated a complete tumor regression. The administration of5 mg/kg of the ChiBR96-ADM conjugate also produced antitumor activityequivalent to >10 LCK with 88% tumor cures and 12% complete tumorregressions. The antitumor activity observed following administration ofChiBR96-ADM conjugates (>10 LCK) was substantially better than thatobserved for optimized adriamycin (1.0 LCK). The CHiBR96-ADM conjugatewas also more potent than optimized adriamycin; that is; the antitumoractivity of the ChiBR96-ADM conjugate tested at a dose of 5 mg/kgequivalent adriamycin was superior to that of adriamycin tested at adose of 8 mg/kg. The non-binding human IgG conjugate (MR=7.16) was notactive against L2987 xenografts when tested at a dose of 10 mg/kgequivalent of adriamycin indicating that the superior activity of theChiBR96-ADM conjugate was due to antigen specific binding of theimmunoconjugate to L2987 tumor cells.

Similar data are presented in FIG. 58. As shown, the ChiBR96-ADMconjugate (MR=5.8) tested at a dose equivalent of 10 mg/kg adriamycinresulted in antitumor activity equivalent to >10 LCK. At this dose, 90%tumor cures and 10% complete tumor regressions were observed. Theadministration of 5 mg/kg of the ChiBR96-ADM conjugate resulted in 4.8LCK with 10% cures, 50% complete and 10% partial tumor regressions. Theantitumor activity of the ChiBR96-ADM conjugate greatly exceeded that ofoptimized adriamycin (1.6 LCK) and, as described above, the ChiBR96-ADMconjugate was more potent than unconjugated adriamycin. The non-bindingIgG-ADM conjugate (MR=7.16) was not active at a dose of 10 mg/kg.

The antitumor activity of various preparation of ChiBR96-ADM conjugatedprepared by the "relaxed" antibody technique and evaluated againstestablished L2987 lung tumor xenograft is presented in Table 15.

                                      TABLE 15                                    __________________________________________________________________________    Antitumor Activity of ChiBR96-ADM Conjugates Against Established                L2987 Human Lung Tumor Xenografts*                                                                  % Tumor                                                 Dose (mg/kg)   Regressions                                                  Conjugate ADM                                                                              Antibody                                                                           Route                                                                            LCK                                                                              PR CR                                                                              Cure                                                                             No. of Mice                                   __________________________________________________________________________    ChiBR96-ADM-6.85                                                                        15 615  ip >10                                                                              10 0 80 10                                               10 410 ip >10 0 0 89 9                                                        8 320 iv >10 0 0 100 9                                                        5 205 iv >10 0 22 78 9                                                       ChiBR96-ADM-4.19 15 980 ip >10 0 11 89 9                                       10 654 ip >10 11 11 66 9                                                      5 327 iv >10 0 11 89 9                                                        2.5 164 iv >10 0 22 78 9                                                     ChiBR96-ADM-6.85 10 410 ip >10 11 11 78 9                                      8 328 iv >10 0 0 100 9                                                        5 205 iv >10 0 11 89 9                                                       ChiBR96-ADM-4.19 10 654 ip >10 0 0 100 9                                       5 327 ip >10 0 0 100 9                                                       ChiBR96-ADM-4.19 10 654 ip >8 0 22 78 9                                        5 327 ip >8 0 11 89 9                                                        ChiBR96-ADM-5.80 10 500 ip >10 0 10 90 10                                      5 250 ip >4.8 10 50 10 10                                                    ChiBR96-ADM-6.82 5 204 iv >10 22 22 55 9                                       2  82 iv 3.5 44 33 0 9                                                        1  41 iv 2.0 0 22 0 9                                                        ChiBR96-ADM-6.82 10 400 ip >5.3 11 11 56 9                                     5 200 ip 4.8 30 10 40 10                                                      2.5 100 ip 2.9 30 0 30 10                                                     1.25  50 ip 1.1 11 0 11 9                                                     0.62  25 ip 0 0 0 0 9                                                         5 200 iv >5.3 10 20 70 10                                                     2.5 100 iv 2.9 22 33 0 9                                                      1.25  50 iv 1.5 11 11 0 9                                                     0.62  25 iv 0.6 0 0 0 9                                                      Adriamycin 8 -- iv 1-1.8 3.6 0 0 55                                         __________________________________________________________________________     *All treatment administered on a q4d × 3 schedule                  

As shown, the antitumor activity of ChiBR96-ADM conjugates is superiorto that of optimized adriamycin and the ChiBR96-ADM conjugates are 6-8fold more potent than unconjugated adriamycin.

The antitumor activity of ChiBR96-ADM conjugates was also evaluatedagainst large (225 mm³) established L2987 tumors (FIG. 59). Theadministration of the ChiBR96-ADM conjugate (MR=6.85) at a dose of 10mg/kg equivalent adriamycin resulted in antitumor activity equivalent to<10 LCK and 70% cures and 30% partial tumor regressions were observed.

The antitumor activity of unconjugated ChiBR96 antibody was evaluatedusing established (50-100 mm³) L2987 human lung tumor xenografts. Asshown in Table 10, ChiBR96 antibody administered at doses of 100, 200 or400 mg/kg was not active against established L2987 tumors. The antitumoractivity of mixtures of ChiBR96 and adriamycin was not different fromthat of adriamycin administered alone. Therefore, the antitumor activityof the CHiBR96-ADM conjugates reflects the efficiency of the conjugateitself rather than a synergistic antitumor effect of antibody andadriamycin.

                  TABLE 16                                                        ______________________________________                                        Antitumor Activity of Adriamycin, ChiBR96, and Mixtures of                      ChiBR96 and Adriamycin Against Established L2987 Human Lung                   Tumor Xenografts                                                                        Dose        Log    % Tumor  No.                                     (mg/kg).sup.a Cell Regressions of                                           Treatment   ADM    ChiBR96  Kill PR  CR  Cure Mice                            ______________________________________                                        Adriamycin  8      --       1.5  0   0   0    9                                 ChiBR96 -- 400 0 0 0 0 8                                                       -- 200 0 0 0 0 8                                                              -- 100 0 0 0 0 8                                                             Adriamycin + 8 400 1.8 11 0 0 9                                               ChiBR96 8 200 1.6 0 0 0 9                                                      8 100 1.9 0 0 0 8                                                          ______________________________________                                         .sup.a Treatment administered iv on a q4d × 3 schedule             

In summary ChiBR96-ADM conjugates demonstrated antigen-specificantitumor activity when evaluated against established L2987 human lungtumors. The antitumor activity of ChiBR96-ADM conjugates was superior tothat of optimized adriamycin, mixtures of ChiBR96 and adriamycin, andequivalent doses of non-binding conjugates. The ChiBR96-ADM conjugateswere approximately 6 fold more potent than unconjugated adriamycin.Cures or complete regressions of established tumors were observed in 50%of animals treated with doses of ≧2.5 mg/kg of ChiBR96-ADM conjugate.

As shown in FIG. 60, ChiBR96-ADM conjugates (MR=7.88) demonstratedantigen-specific antitumor activity against established (745-125 mm³)MCF7 tumors. The activity of ChiBR96-ADM conjugate administered at adose of 5 mg/kg by either the ip or iv route (4.2 LCK) was superior tothat of optimized adriamycin (1.4 LCK) or equivalent doses ofnon-binding IgG conjugate (1.2 LCK). The antitumor activity ofChiBR96-ADM and non-binding IgG-ADM conjugates is summarized in Table17. The MTD of CHiBR96-ADM conjugates like that of free adriamycin islower in the MCF7 model due to the estradiol supplementation requiredfor tumor growth.

                  TABLE 17                                                        ______________________________________                                        Summary of Antitumor Activity of ChiBR96-ADM Thioether                          Conjugates Evaluated Against Established MCF7 Human Breast Tumor             Xenografts                                                                                               Log  % Tumor  No.                                                                            Dose (mg/kg).sup.a  Cell                                                     Regressions of                      Conjugate                                                                              ADM    ChiBR96  Route                                                                              Kill PR  CR  Cures                                                                              Mice                          ______________________________________                                        ChiBR96- 10     350      ip   --.sup.b                                                                           --  --  --   10                              ADM-7.88 5 175 ip 4.2 30 0 0 10                                                5 175 iv 4.2 50 10 0 10                                                      IgG-ADM-7.16 5 225 ip 1.1 0 0 0 10                                             2.5 112 ip 0.6 0 0 0 10                                                      Adriamycin 2.5 112 iv 0.8 0 0 0 10                                             6  0 iv 1.4 0 0 0 10                                                       ______________________________________                                         .sup.a All therapy administered q4d × 3                                 .sup.b 40% lethality occurred at this dose of immunoconjugate            

The antigen-specific antitumor activity and dose response of ChiBR96-ADMconjugates was also evaluated in the RCA human colon carcinoma model.RCA tumors are less senstive to unconjugated adriamycin than are L2987and MCF7 tumors. In addition, as described previously, RCA tumors have alonger tumor volume doubling time than L2987 or MCF7 tumors, are morepoorly vascularized, and the localization of radiolabelled BR64 antibodyis lower in RCA tumors than in L2987 tumors. As shown in FIG. 61, theantitumor activity of the ChiBR96-ADM conjugate (MR=7.88) administeredat a dose of 10 mg/kg was superior to that of adriamycin and anequivalent dose of non-binding IgG conjugates (MR=7.16). As shown inTable 18, the ChiBR96-ADM conjugate tested at a dose of 10 mg/kgproduced antitumor activity equivalent to >3 LCK. At this dose ofChiBR96-ADM conjugate, 89% cures and 11% partial tumor regressionsoccurred. In this experiment, unconjugated adriamycin showed antitumoractivity, equivalent to 0.4 LCK. Thus, in this experiment, the BR96-ADMconjugate produced 89% cures of established tumors whereas unconjugatedadriamycin was inactive.

                  TABLE 18                                                        ______________________________________                                        Summary of Antitumor Activity of ChiBR96-ADM Thioether                          Conjugates Evaluated Against Established RCA Human Colon Tumor                Xenografts                                                                                              Log  % Tumor  No.                                   Dose (mg/kg).sup.a  Cell Regressions of                                     Conjugate                                                                              ADM    ChiBR96  Route                                                                              Kill PR  CR  Cures                                                                              Mice                          ______________________________________                                        ChiBR96- 10     350      ip   >3   11  0   89   9                               ADM-7.88 5 175 ip 0.6 11 22 11 9                                               2.5 85 ip 0.2 0 0 0 9                                                         2.5 85 iv 0.6 11 0 0 9                                                       IgG-ADM-7.16 10 405 ip 0 0 0 0 9                                              Adriamycin 8 0 iv 0.4 0 0 0 9                                               ______________________________________                                         .sup.a All therapy administered q4d × 3                            

In summary, the ChiBR96-ADM conjugate demonstrated antigen-specificantitumor activity in the RCA human colon tumor model. Cures andcomplete regressions of established RCA tumors were observed followingthe administration of ChiBR96-ADM conjugate at doses of 5-10 mg/kg.

The invention has been described with reference to specific examples,materials and data. As one skilled in the art will appreciate, alternatemeans for using or preparing the various aspects of the invention may beavailable. Such alternate means are to be construed as included withinthe intent and spirit of the present invention as defined by thefollowing claims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 4                                           - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Plasmid p - #BR96                                      - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:1:                        - - GCTAGACATA TGGAGGTGCA GCTGGTGGAG TCT       - #                  -      #         33                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Plasmid p - #BR96                                      - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:2:                        - - GCTGTGGAGA CTGGCCTGGT TTCTGCAGGT ACC       - #                  -      #         33                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 720 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Mus muscu - #lus                                       - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..720                                                 - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:3:                        - - ATG GAG GTG CAG CTG GTG GAG TCT GGG GGA GG - #C TTA GTG CAG CCT        GGG       48                                                                    Met Glu Val Gln Leu Val Glu Ser Gly Gly Gl - #y Leu Val Gln Pro Gly            1               5 - #                 10 - #                 15              - - TCC CTG AAA GTC TCC TGT GTA ACC TCT GGA TT - #C ACT TTC AGT GAC TAT           96                                                                       Ser Leu Lys Val Ser Cys Val Thr Ser Gly Ph - #e Thr Phe Ser Asp Tyr                        20     - #             25     - #             30                  - - TAC ATG TGG GTT CGC CAG ACT CCA GAG AAG AG - #G CTG GAG TGG GTC GCA          144                                                                       Tyr Met Trp Val Arg Gln Thr Pro Glu Lys Ar - #g Leu Glu Trp Val Ala                    35         - #         40         - #         45                      - - TAC ATT AGT CAA GGT GAT ATA ACC GAC TAT CC - #A GAC ACT GTA AAG GGT          192                                                                       Tyr Ile Ser Gln Gly Asp Ile Thr Asp Tyr Pr - #o Asp Thr Val Lys Gly                50             - #     55             - #     60                          - - CGA TTC ACC ATC TCC AGA GAC AAT AAG AAC AC - #C CTG TAC CTG CAA ATG          240                                                                       Arg Phe Thr Ile Ser Arg Asp Asn Lys Asn Th - #r Leu Tyr Leu Gln Met            65                 - # 70                 - # 75                 - # 80       - - AGC CGT CTG AAG TCT GAG GAC ACA GCC ATG TA - #T TGT GCA AGA GGC CTG          288                                                                       Ser Arg Leu Lys Ser Glu Asp Thr Ala Met Ty - #r Cys Ala Arg Gly Leu                            85 - #                 90 - #                 95              - - GAC GAC GGG GCC TGG TTT GCT TAC TGG GGC CA - #A GGG ACC ACG ACC GTC          336                                                                       Asp Asp Gly Ala Trp Phe Ala Tyr Trp Gly Gl - #n Gly Thr Thr Thr Val                       100      - #           105      - #           110                  - - TCC TCA GGA TCC GGA GGT GGA GGT TCT GGT GG - #A GGT GGA TCT GGA GGT          384                                                                       Ser Ser Gly Ser Gly Gly Gly Gly Ser Gly Gl - #y Gly Gly Ser Gly Gly                   115          - #       120          - #       125                      - - GGA TCT AAG CTT GAT GTT TTG ATG ACC CAA AT - #T CCA GTC TCC CTG CCT          432                                                                       Gly Ser Lys Leu Asp Val Leu Met Thr Gln Il - #e Pro Val Ser Leu Pro               130              - #   135              - #   140                          - - GTC AGT CTT GGA CAA GCG TCC ATC TCT TGC AG - #A TCT AGT CAG ATC ATT          480                                                                       Val Ser Leu Gly Gln Ala Ser Ile Ser Cys Ar - #g Ser Ser Gln Ile Ile           145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - GTA CAT AAT AAT GGC AAC ACC TTA GAA TGG TA - #C CTG CAG AAA CCA        GGC      528                                                                    Val His Asn Asn Gly Asn Thr Leu Glu Trp Ty - #r Leu Gln Lys Pro Gly                          165  - #               170  - #               175              - - CAG TCT CCA CAG CTC CTG ATC TAC AAA GTT AA - #C CGA TTT TCT GGG GTC          576                                                                       Gln Ser Pro Gln Leu Leu Ile Tyr Lys Val As - #n Arg Phe Ser Gly Val                       180      - #           185      - #           190                  - - CCA GAC AGG TTC AGC GGC AGT GGA TCA GGG AC - #A GAT TTC CTC AAG ATC          624                                                                       Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Th - #r Asp Phe Leu Lys Ile                   195          - #       200          - #       205                      - - AGC AGA GTG GAG GCT GAG GAT CTG GGA GTT TA - #T TAC TGC TTT CAA GTT          672                                                                       Ser Arg Val Glu Ala Glu Asp Leu Gly Val Ty - #r Tyr Cys Phe Gln Val               210              - #   215              - #   220                          - - CAT GTT CCA TTC ACG TTC GGC TCG GGG ACC AA - #G CTG GAG ATC AAA CGC          720                                                                       His Val Pro Phe Thr Phe Gly Ser Gly Thr Ly - #s Leu Glu Ile Lys Arg           225                 2 - #30                 2 - #35                 2 -      #40                                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 240 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -           (xi) SEQUENCE DESCRIPTION: - # SEQ ID NO:4:                     - - Met Glu Val Gln Leu Val Glu Ser Gly Gly Gl - #y Leu Val Gln Pro        Gly                                                                               1               5 - #                 10 - #                 15             - - Ser Leu Lys Val Ser Cys Val Thr Ser Gly Ph - #e Thr Phe Ser Asp Tyr                   20     - #             25     - #             30                  - - Tyr Met Trp Val Arg Gln Thr Pro Glu Lys Ar - #g Leu Glu Trp Val Ala               35         - #         40         - #         45                      - - Tyr Ile Ser Gln Gly Asp Ile Thr Asp Tyr Pr - #o Asp Thr Val Lys Gly           50             - #     55             - #     60                          - - Arg Phe Thr Ile Ser Arg Asp Asn Lys Asn Th - #r Leu Tyr Leu Gln Met       65                 - # 70                 - # 75                 - # 80       - - Ser Arg Leu Lys Ser Glu Asp Thr Ala Met Ty - #r Cys Ala Arg Gly Leu                       85 - #                 90 - #                 95              - - Asp Asp Gly Ala Trp Phe Ala Tyr Trp Gly Gl - #n Gly Thr Thr Thr Val                  100      - #           105      - #           110                  - - Ser Ser Gly Ser Gly Gly Gly Gly Ser Gly Gl - #y Gly Gly Ser Gly Gly              115          - #       120          - #       125                      - - Gly Ser Lys Leu Asp Val Leu Met Thr Gln Il - #e Pro Val Ser Leu Pro          130              - #   135              - #   140                          - - Val Ser Leu Gly Gln Ala Ser Ile Ser Cys Ar - #g Ser Ser Gln Ile Ile      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Val His Asn Asn Gly Asn Thr Leu Glu Trp Ty - #r Leu Gln Lys Pro        Gly                                                                                             165  - #               170  - #               175             - - Gln Ser Pro Gln Leu Leu Ile Tyr Lys Val As - #n Arg Phe Ser Gly Val                  180      - #           185      - #           190                  - - Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Th - #r Asp Phe Leu Lys Ile              195          - #       200          - #       205                      - - Ser Arg Val Glu Ala Glu Asp Leu Gly Val Ty - #r Tyr Cys Phe Gln Val          210              - #   215              - #   220                          - - His Val Pro Phe Thr Phe Gly Ser Gly Thr Ly - #s Leu Glu Ile Lys Arg      225                 2 - #30                 2 - #35                 2 -      #40                                                                           __________________________________________________________________________

What is claimed is:
 1. An immunoconjugate comprising a moleculecontaining the antigen-binding region of the BR96 monoclonal antibody(ATCC Accession No. 10036) joined to a therapeutic agent.
 2. Theimmunoconjugate of claim 1, wherein the molecule comprises BR96monoclonal antibody (ATCC Accession No. 10036) or antigen bindingfragments thereof.
 3. The immunoconjugate of claim 2, wherein thefragments are selected from the group consisting of Fv, F(ab') andF(ab')₂ fragments.
 4. The immunoconjugate of claim 1, wherein themolecule comprises chimeric human/murine BR96 antibody (ATCC AccessionNo. 100460) or antigen binding fragments thereof.
 5. The immunoconjugateof claim 4, wherein the fragments are selected from the group consistingof Fv, F(ab)' and F(ab')₂ fragments.
 6. A method for selectively killingtumor cells expressing the antigen that immunospecifically binds to BR96monoclonal antibody (ATCC Accession No. 10036) comprising reacting theimmunoconjugate of claim 1 with said tumor cells.
 7. A bispecificantibody with a binding specificity for two different antigens, one ofthe antigens being that with which the monoclonal antibody of claim 1binds.
 8. The bispecific antibody of claim 7, wherein one of theantigens comprises a variant of Le^(y) determinant which includes anepitopic site containing fucose α1-3.
 9. The immunoconjugate of claim 1,wherein the therapeutic agent is an anti-tumor drug, a cytotoxin, aradioactive agent, a second antibody or an enzyme.
 10. Theimmunoconjugate of claim 9, wherein the cytotoxin is a ribosome bindingtoxin.
 11. The immunoconjugate of claim 10, wherein the ribosome bindingtoxin is ricin A.
 12. The immunoconjugate of claim 9, wherein thecytotoxin is an exotoxin.
 13. The immunoconjugate of claim 12, whereinthe exotoxin is Pseudomonas exotoxin A.
 14. The immunoconjugate of claim12 or 13, wherein the exotoxin is truncated to remove the cell-bindingdomain.
 15. The immunoconjugate of claim 14, wherein the amino terminusof the exotoxin has been modified to include a lysine amino acidresidue.
 16. A method for treating a subject suffering from a malignantdisease characterized by cells having the BR96 antigen on the cellsurface which comprises administering to the subject an effective amountof an immunoconjugate of claim 1 such that the immunoconjugate binds theBR96 antigen and kills said cells thereby treating the subject.
 17. Apharmaceutical composition useful in killing human cells expressing theBR96 antigen on the cell surface comprising a pharmaceutically effectiveamount of the immunoconjugate of claim 1, and a pharmaceuticallyacceptable carrier.
 18. An immunoconjugate of claim 1 which is asingle-chain immunoconjugate.
 19. The single chain immunoconjugate ofclaim 18, wherein the therapeutic agent is an exotoxin.
 20. The singlechain immunoconjugate of claim 19, wherein the exotoxin is Pseudomonasexotoxin A.
 21. The single chain immunoconjugate of claim 19 or 20,wherein the exotoxin has been modified to remove the cell-bindingdomain.
 22. An immunoconjugate comprising the BR96 monoclonal antibody(ATCC Accession No. 10036) or antigen binding fragments thereof joinedto Pseudomonas exotoxin A.
 23. An immunoconjugate comprising chimerichuman/murine BR96 antibody (ATCC Accession No. 100460) joined toPseudomonas exotoxin A.
 24. An immunoconjugate comprising a Fab'fragment of chimeric human/murine BR96 antibody (ATCC Accession No.100460) joined to Pseudomonas exotoxin A.
 25. An immunoconjugatecomprising a F(ab)₂ fragment of chimeric human/murine BR96 antibody(ATCC Accession No. 100460) joined to Pseudomonas exotoxin A.
 26. Arecombinant single-chain immunotoxin molecule comprising a cloned heavychain Fv portion of the BR96 monoclonal antibody (ATCC Accession No.10036) and a cloned light chain Fv portion of the BR96 monoclonalantibody joined to a cytotoxic agent.
 27. The immunotoxin molecule ofclaim 26, wherein the cytotoxic agent is an exotoxin.
 28. Theimmunotoxin molecule of claim 27, wherein the exotoxin is Pseudomonasexotoxin A.
 29. The immunotoxin molecule of claim 27 or 28, wherein theexotoxin has been modified to remove the cell-binding domain.
 30. Amethod for using the immunotoxin molecule of claim 26, to selectivelykill tumor cells expressing the antigen that specifically binds to BR96monoclonal antibody (ATCC Accession No. 10036) comprising contactingsaid tumor cells with an effective amount of the immunotoxin moleculeeffective to kill said tumor cells.
 31. A method for selectively killingtumor cells expressing the antigen that specifically binds to BR96monoclonal antibody (ATCC Accession No. 10036) comprising contactingsaid tumor cells with an amount of the immunotoxin molecule of claim 26for a time sufficient to kill said cells.
 32. A single-chainimmunoconjugate comprising a Fv fragment of BR96 antibody (ATCCAccession No. 10036) joined to Pseudomonas exotoxin A.
 33. Asingle-chain immunoconjugate comprising a Fv fragment of chimerichuman/murine BR96 antibody (ATCC Accession No. 100460) joined toPseudomonas exotoxin A.
 34. A method for imaging carcinoma characterizedby cells having the BR96 antigen on the cell surface comprisingadministering to a patient intravenously the BR96 monoclonal antibody(ATCC Accession No. 10036) in an amount effective for detection of thecarcinoma, allowing the antibody to bind to carcinoma cells and tolocalize to the site of carcinoma cells and detecting said antibodybound to the carcinoma cells.
 35. The method of claim 34, wherein saidantibody is labeled so as to directly or indirectly produce a detectablesignal with a label selected from the group consisting of a radiolabel,an enzyme, a chromophore, and a fluorescer.